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Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ Microscopy studies on uncultivated magnetotactic bacteria T. S. Silveira, J. L. Martins, K. T. Silva, F. Abreu & U. Lins∗ Instituto de Microbiologia Professor Paulo de Góes, Universidade Federal do Rio de Janeiro, 21941-590, Rio de Janeiro, RJ, Brazil Magnetotactic bacteria are characterized by the magnetosome, a membrane-bound, nano-sized organelle containing a magnetic crystal. This organelle is believed to help the bacteria to navigate along the geomagnetic field lines and find a preferred habitat for survival. The magnetosome is a biomineral structure highly controlled by the bacteria, composed of either magnetite (Fe3O4) or greigite (Fe3S4), and arranged in one or more linear chains in the cell. Most magnetotactic bacteria are not cultivated and studies rely on environmental samples, where microscopy techniques are powerful tools for the structural characterization of the bacteria and their magnetosomes. Here, we show examples of the application of microscopy techniques to the study of uncultivated magnetotactic bacteria. Keywords magnetosome; magnetotactic bacteria; magnetotaxis; biominerals. 1. Introduction Magnetotactic bacteria (MB) are prokaryotes that align and migrate along the lines of a magnetic field. All MB are associated to the domain Bacteria, are gram-negative and motile by flagella. MB generally live in aquatic habitats and respire microaerobically or anaerobically. The hallmark of all magnetotactic bacteria is the unique ability to produce magnetosomes. Magnetosomes are organelles that consist of a magnetic nanoparticle enveloped by a membrane. Each MB species or strain can produce only one type of magnetosome of a specific shape, size and composition [1] with very few exceptions [2, 3]. The production of magnetosomes is encoded and regulated at gene level [1]. The magnetosome membrane is supposed to originate from the cytoplasmic membrane based on its lipid and protein composition [4] at least in the cultivated Magnetospirillum species [5]. Some recent studies suggest they are structurally contiguous with the plasma membrane, like invaginations, in Magnetospirillum magneticum [6]. The magnetosome membrane contains exclusive proteins that are thought to have a crucial importance in the magnetic particle nucleation, growth and arrangement [7]. Advances in this subject depend on the establishment of a genetic and biochemical model, which is gradually been achieved with the increasing characterization of some cultivated species. Two types of magnetic minerals are found in magnetosomes: the iron oxide magnetite (Fe3O4) and the iron sulfide greigite (Fe3S4). The crystalline part of the magnetosome has been extensively studied with microscopy, especially high resolution transmission electron microscopy (HRTEM). Bacterial magnetite typically has cube {100}, octahedron {111}, and dodecahedron {110} faces in crystals that are commonly elongated [8] or bullet-shaped [9, 10, 11]. In contrast most greigite magnetosomes occur in parallelepiped or cuboctahedral with irregular border [12]. A good review on magnetosome crystallographic properties can be found in [13]. Other non-magnetic minerals, like tetragonal or cubic FeS, were reported as precursors of greigite [14]. Magnetite-containing MB can be collected in freshwater, brackish or marine environments, whereas greigite-containing MB seems to be restricted to anoxic environments [14]. The number of magnetosomes varies from a few to thousands per cell [15]. The magnetosomes are generally in the range of 35-120 nm [1], which corresponds to the single-domain size range for magnetite or greigite. However, magnetosomes up to 250 nm were reported [15, 16]. ∗ Corresponding author: e-mail: [email protected], Phone: +55212562-6738 111 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ The magnetosomes form chains within the cell, which impart the cell body a magnetic moment that overcomes the viscous drag of the environment where the bacteria live. This allows the efficient orientation of the cell to the magnetic field. When magnetosomes are arranged in a linear chain within the bacterial cell, the total magnetic moment is the highest for that assembly [17, 18]. Biophysical arguments [19] indicate that supporting structures are necessary to prevent the chains from collapsing into a disordered clump of particles and a cytoskeleton connection between the cell envelope and the magnetosome chains was proposed for the efficient transfer of torque to the cell body [20, 21]. There are at least two hypotheses to explain how the magnetic torque is transmitted from magnetosomes to the cell. In cocci, the magnetotactic repulsion between two or more magnetosome chains drives them apart from each other. As a consequence, the chains touch the cytoplasmic membrane. In this case, the torque generated by a situation of field modification is efficiently transferred from the chain to the whole cell by mechanical contact [22, 23]. The second hypothesis proposes the existence of specific molecules to anchor magnetosomes in the proper place in the cell [20, 21]. Recently evidences of the presence of such molecules have been described in Magnetospirillum species [6, 24, 25]. The model of passive orientation of MB along the lines of a magnetic field combined with the active migration because of the flagella, the so-called magnetotaxis, is maintained until today. Magnetotaxis is considered an advantage in finding the optimum position in the environment because MB would have the geomagnetic poles as fixed references to swim, unlike other bacteria. So, if a magnetotactic bacterium is moved away from its niche, it would use the lines of a magnetic field to return to its preferred habitat, whereas other bacteria would randomly swim until it reached the habitat, thus, expending more time and, probably, more energy. A major improvement in the magnetotaxis model was the combination of magnetotaxis and aerotaxis, the so-called magneto-aerotaxis. Based on experimental findings of MC-1, a cultivated coccus [26], the magneto-aerotaxis model states that MB can be either in an oxidized or reduced state. Considering that MB are in a vertical gradient of oxygen, with the saturated O2 waters in the upper layers, they swim downwards to reach an optimum position (remember: MB are microaerophiles or anaerobes). MB do it by rotating their flagella counterclockwise, because they are in the oxidized state. However in the reduced state, their flagella would rotate clockwise and the bacteria should move upwards, towards the preferred oxygenated conditions. In this model, MB still benefits from having a fixed axis for swimming and do not need to “run and tumble” as non-magnetotactic bacteria. There is evidence that magneto-aerotaxis cannot explain the behavior of all MB populations [27], but other models to extend or substitute magneto-aerotaxis were not suggested yet. The vertical chemical gradient used in experiments for the magneto-aerotaxis was verified in some MB natural habitats, generally associated to an opposite sulfide gradient. Carefull measurements showed that MB occur in the oxic-anoxic interface (OAI) or slightly above or below this transition zone [28, 29]. The OAI can be established in the water column or in the first centimeters of the sediment. Iron gradients can also influence their distribution and peaks of dissolved and particulated iron can be related to their presence [28]. Other gradients are not known to influence MB local distribution, but sources of carbon are thought to be important to their positioning [30]. Cultivated strains indicate that short chain fatty acids, tricarboxylic acid cycle intermediates and acetate, can be used as carbon sources. These molecules are typical products of fermentation and are generally present in the suboxic zone [30]. MB are distributed worldwide and the most common morphotypes are spirilla, cocci, rods, vibrios and the unusual multicellular form, the multicellular magnetotactic prokaryote (MMP). MB differ in flagella arrangement, placed monotrichously or lophotrichously in the cells; cytoplasmic contents, like carbon, sulfur or phosphorous inclusions; and magnetosome number, shape, size and composition. There are sampling sites where only one morphotype can be recovered [29, 31] but this is not common. There is no consistent relationship among a morphotype, a phylotype and/or the mineral produced. MB are related to the alphaproteobacteria, such as the genus Magnetospirillum, MC-1 and MV-1 [32]; others are related to the deltaproteobacteria, like some uncultured multicellular forms, the recently named Candidatus Magnetoglobus multicellularis [29] and Desulfovibrio magneticus; an uncultured rod is affiliated to gammaproteobacteria group [33]; and the Nitrospira phylum which includes an uncultured MB, the tentatively named “Magnetobacterium bavaricum” [15]. Few MB were phylogenetically studied so far 112 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ and not even a fraction of their potential habitats was explored. Therefore, the phylogenetic diversity is largely unknown. As few species or strains can be cultured nowadays, most of the studies rely on uncultured MB. But how to detect and recognize MB in these samples? Although simple, the question prompts for criteria to identify the magnetotactic behavior and to distinguish the magnetosomes from other commonly found inclusion in prokaryotes. Because the currently available culture media are not selective enough to guarantee retrieving only magnetotactic bacteria, light and electron microscopy are still the most important and straightforward culture-independent techniques to determine if a bacterium is magnetotactic. Here, we discuss some uses of microscopy to detect uncultured MB. Two examples of MB are illustrated. 2. Light microscopy Light microscopy is the most used technique to detect MB in environmental samples. Water and sediment samples can be observed between a slide and coverslip or in a hanging drop. If MB are expected to be in the sediment, water and sediment from the sampling site is placed on a slide and observed preferably with a long working distance objective. At that time, one must look for swimming bacteria and response to magnetic field. The discovery of MB by R. Blakemore was based on the observation of the motility of bacteria in the local magnetic field of his laboratory [34]. Nowadays, an ordinary magnet is placed near the slide for few minutes. The light microscopy observation leads to a first recognition of MB and their magneto-aerotactic behavior. Under a magnetic field lines, most bacteria will accumulate at one edge of the drop and the bacteria are said to have polar magnetotactic behaviour. If the magnetic field lines define only the axis of swimming, changes in the magnetic field direction will cause bacteria only to rotate, not accumulate at the edge of the drop [26]. This behaviour is known as axial magnetotaxis and it is far less common than polar magnetotaxis [26]. Light microscopy lacks the resolution to individualize each magnetosome, but an accurate observation in bright field can reveal the magnetosome chain as a dark dot or a small segment within the cells (Fig. 1a). Some MB contain other inclusions that can be seen by light microscopy. Sulphur inclusions appear as birefringent spheres when imaged with phase contrast microscopy and colourless inclusions are easily observed by differential interferential contrast. Flagella bundles can be observed with dark field microscopy. Fluorescence in situ hybridization (FISH) has been used to study the population structure and function. The rRNA-targeted probes used in this method allow the identification and quantification of bacteria [35]. The association of FISH with fluorescence microscopy is frequently used to access the phylogenetic relationships between MB because it overcomes the difficulty of cultivation and confirms the origin of the sequences retrieved from magnetic enrichments (Figs. 2b-d). Individual magnetosome chains were observed by reflectance confocal laser scanning microscopy [36]. This technique allows the rapid detection and location of magnetosomes even in small quantities of material. Details of magnetosome size or shape cannot be observed with this technique. Confocal laser scanning microscopy was also used to show the arrangement of living cells in “Candidatus M. multicelularis” using a fluorescent lipophilic dye [37]. It has also been used to demonstrate that all cells within a MMP belong to the same species [38] and to measure the acidic nature of grazing protozoa that ingested MB [39]. 3. Transmission electron microscopy Transmission electron microscope (TEM) is the technique of choice for observing individual magnetosomes and to absolutely classify a bacterium as capable of producing magnetosomes. The simplest preparation method consists of placing a suspension of bacteria on a Formvar or carbon coated grid and checking for magnetosome positioning in chains within the cell (Fig. 3a). Magnetosomes can occur in single, double or multiple chains, peripherally or along the major axis of the cell. Clusters of magnetosomes arranged in a disordered pattern are occasionally observed. This configuration is interpreted as a consequence of the disorganization caused by the loss of chain stability. The magnetosome shape can be determined on TEM whole-mount cell preparation or ultra-thin sections. 113 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ TEM also allows counting and measuring of the magnetosomes. Magnetosome size distribution is used as a marker of the biological origin of the crystals [40]. Other intracellular structures such as carbon, sulphur, or phosphorous inclusions are observed [41]. Electron-dense inclusions can be sometimes difficult to distinguish from magnetosomes, because of the similar morphology and size. X-ray microanalysis is the first approach to elucidate the composition of the mineral phase of magnetosomes and to check for the purity of the crystal. A typical spectrum shows peaks for oxygen and iron or sulphur and iron, which indicates that magnetite (Fe3O4) or greigite (Fe3S4), respectively, are present (Fig. 3b). A conclusive classification needs electron diffraction analysis or HRTEM, two techniques that can differentiate among minerals and their possible precursors. Recently, several authors claimed to have isolated new strains of MB [42, 43]. However, the authors fail to demonstrate that the electron-dense structures are magnetosomes. The individual “crystal” morphology is difficult to see and unusually irregular for magnetosomes. The irregular inclusions within the cytoplasm of the isolated strains are more likely amorphous inclusions bodies, instead of magnetosomes, which are not irregular particles, but ordered crystalline nanostructures. Even greigite pleiomorphic magnetosomes show a high degree of structural organization. As no crystallographic data on the “magnetosomes” (electron diffraction or HRTEM) is presented it is difficult to conclude that the newly described structures are magnetosomes. Recently, non-crystalline magnetic inclusions were reported in prokaryotic cells [44], but they cannot be considered true magnetosomes. Negative staining contributes to the observation of flagella (Fig. 2i), which are fragile structures frequently lost during simple preparation, probably because of mechanical stress. Differently from other bacteria, MB produce short flagella, with filaments less than one helix turn [45]. The short flagella force the bacterium body to rotate around its axis while it swims. Under the geomagnetic field, the beating of the short flagella causes a misalignment of the trajectories [46] that enables the cell to change the trajectory direction if it hits an obstacle in the sediment. Ultra-thin sections are used to observe distinct internal details of the cell, including magnetosomes. The magnetosome membrane is recognized to have a critical importance in biomineralization [1]. However, a trilaminar appearance of membranes, typical of osmium stained preparations, is difficult to be visualized, mainly because of the diffraction conditions of the crystal. Not all magnetosomes are believed to be enveloped by lipid membranes, but protein [22] or a matrix of unknown composition [47]. Freeze-fracture and freeze-etching have been used to study the properties of the magnetosome membranes in cultivated species [24] and the surface organization of the cells [48, 49]. Structures connecting the cell surface of Magnetospirillum cells to the magnetosome chains [24] as well as numerous membranes vesicles were described with these cryotechniques [48]. Uncultured magnetotactic cocci have a complex surface composed of microdomains of proteins (S-layers) which covers the cell (Fig. 1e) and the flagella tufts were observed to emerge from a depression in the cell surface [49]. The association of gold labelling and phylogenetic affiliation with in situ hybridization in TEM identified uncultured cocci isolated from Itaipu lagoon, in Brazil (Fig. 1f). Polyribonucleotides probes were labelled with digoxigenin or fluorescein and detected by immunolabelling with anti-fluorescein and anti-digoxigenin antibodies labelled with 10 and 15 nm gold-particles. This is possibly the only technique that can associate the phylogenetic positioning of a bacterium with its magnetosome morphology [50]. 4. Scanning electron microscopy Scanning electron microscopy (SEM) is a powerful technique for studying biomineralization products. Their surface and crystallographic features can be studied. In unicellular MB, SEM has limited use as most bacteria are morphologically simple. An exception is the MMP which is morphologically unique among prokaryotes and can be easily distinguished in a sample because of its “mulberry” appearance in SEM (Figs 2e-g) and in light microscopy (Fig. 2a). Estimated cell numbers and volumes were key parameters for suggesting an exclusively multicellular life cycle for this microorganism and SEM images greatly illustrated this hypothesis [31]. 114 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ Isolated magnetosomes have been imaged with field emission scanning electron microscopy (Fig. 1d) and the results compared to scanning probe microscopy [51] and energy-filtered TEM of platinum shadowed isolated crystals [52]. All techniques are useful for studying the surface of magnetosome and other biominerals. Depending on the treatment used to isolate the magnetosomes, some organic material remains (probably membrane proteins and lipids) attached to the crystals and the surfaces seem irregular. 5. Illustrated examples 5.1 Cocci from Itaipu lagon Itaipu Lagoon (43o04’W, 22o57’S), is located on the coast of Brazil north of Rio de Janeiro. This lagoon communicates with the sea through an artificial channel. At least four coccoid morphotypes with magnetite magnetosomes, Itaipu-1, -2, -3 and -4 [50], a rarely observed rod-shaped bacterium [53], and MMPs [3], occur in the lagoon. Itaipu-1, the largest coccoid microorganism, contains two chains of magnetosomes. The magnetosome crystals are the largest-volume magnetosome crystals yet reported with lengths up to 250 nm and width-to-length ratios of about 0.9 [16]. Itaipu-2 and -4 are smaller cocci containing magnetosome crystals that are smaller, but with similar projected shapes to those in Itaipu-1. Itaipu-3 has magnetosome crystals with lengths up to 120 nm and prominent corner facets. Whereas magnetosomes in most magnetotactic bacteria, including Itaipu-3, are permanent single-magnetic domains, it has been previously reported that the large magnetosomes in Itaipu-1 are metastable, singlemagnetic domains [18]. TEM microdiffraction combined with HRTEM was used to provide information on the crystallography of individual magnetosomes isolated from the cells [16]. It is remarkable that the crystals in the chain of Itaipu-1 remain in a chainlike disposition after isolation (Fig. 1c). The crystals align with their [111] elongation axes parallel to the chain direction and all in the [1-10] zone, and are also ordered with like corner faces of adjacent crystals facing each other [16]. This particular feature may be used as a potential biomarker for magnetite formation in MB. Electron spectroscopic imaging was used to directly image magnetosome size and distribution within Itaipu cocci (Fig 1b) as well as other uncultured bacteria [9]. With this technique, the magnetosome arrangement and the disposition of flagella were associated with unique clarity. Also, the magnetosome substructure was analyzed. Distortions in magnetosomes caused by strong strain magnetic fields and twinned crystals were also observed [9]. Electron spectroscopic imaging is a powerful and efficient tool for studying the diversity of MB and magnetosomes biomineralization because of the minimal requirements for specimen preparation, and because of the acquisition of detailed information through the simultaneous visualization of bacterial morphology, crystal morphology, and chain alignment. This would be difficult to be achieved, at least with the same efforts, by other microscopic techniques. One major disadvantage is that it consists of a rather specialized technique which requires sophisticated instrumentation. Electron spectroscopic images were used to calculate high-resolution maps of the elements of magnetosomes in plastic resin embedded and ultra-thin sectioned Itaipu cocci. Iron (Fig. 3c) and oxygen (Fig. 3d) maps were used to identify the crystals of the magnetosomes as containing magnetite. Electron energy-loss spectroscopy of the magnetosomes confirmed the oxygen and iron elements (Fig. 3e). Similarly sulfur maps and spectra can be produced for greigite magnetosomes. 5.2 Candidatus Magnetoglobus multicelularis Recently, an intriguing MMP has been described as “Candidatus Magnetoglobus multicelularis” [29]. This curious multicellular form of MB (Fig. 2a) is found in a hypersaline lagoon in Rio de Janeiro and showed many characteristics that are unique among prokaryotes. Phylogenetic analysis of “Candidatus Magnetoglobus multicelularis” and FISH associated this microorganism to the deltaproteobacterium group of Bacteria (Figs. 2b and 2c). “Candidatus Magnetoglobus multicelularis” have an unusual multicellular life cycle, cells precisely organized, polarized and unable to live individually. It is 115 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ composed of gram-negative bacterial cells which are genetically identical and arranged side by side in a sphere that moves as a unity in straight or helicoidal trajectories. Each cell faces both the external environment and an acellular compartment in the middle of microorganism (Fig. 2h). This acellular compartment is maintained during the multicellular life cycle of “Candidatus Magnetoglobus multicelularis”, in which one microorganism (a) grows, (b) doubles the volume and the number of constituent cells, (c) reorganizes the cells and then (d) divides into two new and identical microorganisms. On the surface of the microorganism, cells are covered by a capsule and numerous flagella (Fig 2i). The flagella are responsible for its coordinated complex movement, including rotation and the so-called escape motility. “Candidatus Magnetoglobus multicelularis” contain thousands of magnetosomes (Fig. 3a) composed of iron sulfide (Fig. 3b). The magnetosomes are enveloped by a membrane and are distributed in planar groups near the periphery of the microorganism. Unfortunately, “Candidatus Magnetoglobus multicelularis” is an uncultured microorganism and many interesting questions involving multicellularity and magnetosome formation are still unsolved. Recently, we demonstrated that grazing ciliates can ingest this microorganism (Fig. 2j) and dissolve the iron sulfide magnetosomes in the ciliate acid digestive vacuoles [39]. We do not know the impact of the magnetosomes digestion by the ciliates in the environment. Previous estimates have shown that marine MB can reach populations densities of 104 cells/ml [54]. The large amount of iron concentrated in magnetosomes (10-13 to 10-15 g iron per cell), considering their estimated population density, indicates that nanomolar concentrations of iron may be sequestered in the biomass. The ingestion of “Candidatus Magnetoglobus multicelularis” by ciliates shows the possibility of recycling of the iron trapped within magnetosomes. Thus, depending of the rate consumption part of the iron trapped within the magnetosomes is recycled to the environment in a more soluble form. Each microorganism contains about 30 cells with up to 100 magnetosomes, which adds up to 3000 magnetosomes per microorganism. For an average magnetosome size of 100nm, which results in a cubic volume of 106nm3, an estimated total amount of 1.2 x 10-11g of greigite per microorganism (greigite density = 4.1g/cm3) [55] can be solubilized within the ciliates and eventually returned to the environment. For a population of 6.2 x 103 “Candidatus Magnetoglobus multicelularis”/ml in the sediment, nanomolar concentrations of iron can be recycled. 6. Perspectives Magnetotactic bacteria and magnetosomes are subject of an active and interdisciplinary field of research which includes not only microscopy but other topics such as cell biology, geomicrobiology, palaeomagnetism, biomineralization, biophysics and biochemistry to name a few. New and exciting findings are constantly being reported on these microorganisms. Of course, these breakthroughs do not always include, but usually benefit from all aspects of microscopy. New directions will come that will inspire research groups in this highly multidisciplinary field of microbiological research; future developments in light and electron microscopy will be certainly very helpful. Acknowledgements: U.L thanks Prof. Marcos Farina and Carolina N. Keim for previous collaborative work. This work was supported by CNPq, CAPES, FAPERJ (Pronex) Brazilian agencies. 116 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ Fig 1. Microscopy of Itaipu cocci. a) Bright-field image where cocci with magnetosome chains are observed. The inset shows the magnetosome chains (arrow), bar: 5µm and 500 nm in the inset; b) Electron spectroscopic image showing the magnetosomes with Itaipu-4 morphotype, bar: 500 nm. c) Transmission electron microscopy image of isolated magnetosomes. The largest particles come from Itaipu-1 morphotype, bar: 600 nm. d) Scanning electron microscopy image of isolated magnetosomes, bar: 200 nm. e) Freezing-etching image of a Itaipu coccus. Note the magnetosome chains and the domains on the surface of the cell., bar: 500 nm, f) In situ hybridization of an ultra-thin section of Itaipu-1 with a specific 16S rDNA -digoxigenin (DIG) probe labelled with anti-DIG antibody-gold particle, bar: 300 nm. 117 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ Fig. 2 Microscopy of “Candidatus Magnetoglobus multicellularis”. a) Nomarski interferential contrast image of several microorganisms, bar: 5 µm; b-d) Fluorescence micrographs of DAPI-stained microorganisms (b), and fluorescent in situ hybridization with the probes for multicellular forms of MB: MMP [56] (c) and “Candidatus Magnetoglobus multicellularis” [29] (d), bar: 5 µm. e-f) SEM of three microorganisms, bar: 1 µm. h) Ultra-thin section of “Candidatus Magnetoglobus multicellularis”, bar: 1µm. i) TEM of a whole-mount microorganism showing its short flagella (arrow), bar: 250 nm; j) TEM image of a degraded “Candidatus Magnetoglobus multicellularis” ingested by a ciliate. The inset shows a high magnification image where magnetosomes can be seen, bars: 2 µm and 50 nm in the inset. 118 Modern Research and Educational Topics in Microscopy. ©FORMATEX 2007 A. Méndez-Vilas and J. Díaz (Eds.) _______________________________________________________________________________________________ Fig. 3: a) TEM image of a whole-mount “Candidatus Magnetoglobus multicellularis” where numerous cells are observed. A “Candidatus Magnetoglobus multicellularis” can contain more than 5000 magnetosomes. The inset shows chains of magnetosomes (arrowhead) within a cell, bar: 2µm and 300 nm in the inset; b) X-ray microanalysis spectrum of a magnetosome (asterisk) from a “Candidatus Magnetoglobus multicellularis”. Note the presence of sulphur and iron, indicating iron sulphides as the crystalline part of the organelle. All other elements come from the cytoplasm or the grid bar (Cu); c-d) Elemental mapping of magnetosomes from a thin-sectioned Itaipu coccus calculated by the three window power-low method. 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