Spore Structure Research Articles

The sps Genes Encode an Original Legionaminic Acid Pathway Required for Crust Assembly in Bacillus subtilis.

The crust is the outermost spore layer of most Bacillus strains devoid of an exosporium. This outermost layer, composed of both proteins and carbohydrates, plays a major role in the adhesion and spreading of spores into the environment. Recent studies have identified several crust proteins and have provided insights about their organization at the spore surface. However, although carbohydrates are known to participate in adhesion, little is known about their composition, structure, and localization. In this study, we showed that the spore surface of Bacillus subtilis is covered with legionaminic acid (Leg), a nine-carbon backbone nonulosonic acid known to decorate the flagellin of the human pathogens Helicobacter pylori and Campylobacter jejuni We demonstrated that the spsC, spsD, spsE, spsG, and spsM genes of Bacillus subtilis are required for Leg biosynthesis during sporulation, while the spsF gene is required for Leg transfer from the mother cell to the surface of the forespore. We also characterized the activity of SpsM and highlighted an original Leg biosynthesis pathway in B. subtilis Finally, we demonstrated that Leg is required for the assembly of the crust around the spores, and we showed that in the absence of Leg, spores were more adherent to stainless steel probably because of their reduced hydrophilicity and charge.IMPORTANCEBacillus species are a major economic and food safety concern of the food industry because of their food spoilage-causing capability and persistence. Their persistence is mainly due to their ability to form highly resistant spores adhering to the surfaces of industrial equipment. Spores of the Bacillus subtilis group are surrounded by the crust, a superficial layer which plays a key role in their adhesion properties. However, knowledge of the composition and structure of this layer remains incomplete. Here, for the first time, we identified a nonulosonic acid (Leg) at the surfaces of bacterial spores (B. subtilis). We uncovered a novel Leg biosynthesis pathway, and we demonstrated that Leg is required for proper crust assembly. This work contributes to the description of the structure and composition of Bacillus spores which has been under way for decades, and it provides keys to understanding the importance of carbohydrates in Bacillus adhesion and persistence in the food industry.

Characterization of fungal flora associated with sternorrhyncha insects of cotton plants

In this study, 22 different fungal isolates associated with naturally dead insects of order Hemiptera including Bemisia tabaci, Aphis gossypii and Phenacoccus solenopsis were isolated from three different agroecological zones namely hot arid, cotton and central mixed zone. Fungal species were characterized morphologically on the basis of colony color, shape and spore structures through using different dichotomous keys. As a result fungi belonging to different species of Aspergillus, Acremonium, Beauveria, Cladosporium, Alternaria, Clonostachys, Penicillium, Trichoderma, Fusarium, Metarhizium and Verticillium were identified. All isolates of individual species were identical with respect to morphological differentiation. Principal component analysis (PCA) indicates strong correlation between Verticillium lecanii and Aspergillus fumigatus with respect to their isolation source (insect host). However, based on PCA, an uneven distribution of entomopathogenic fungi in different insect hosts is evident. Phylogenetic analysis revealed that Aspergillus fumigatus and A. nidulans are more closely related to different species of Penicillium, while Aspergillus oryzae and A. flavus formed separate clades, whereas different species of Alternaria, Cladosporium and Fusarium formed separate clades. Thus, the information generated will add to our understanding of the insect associated fungal communities for future development of potential biocontrol agents and their application as a vital component of sustainable agriculture.

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation, Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus

Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum, temperature extremes and a lack of water and nutrients. A wide variety of terrestrial microorganisms, including those counted amongst the most ancient inhabitants of Earth, can cope with high levels of salinity, extreme temperatures, desiccation and high levels of radiation. Key among these are the haloarchaea, considered particularly relevant for astrobiological studies due to their ability to thrive in hypersaline environments. In this study, a novel haloarchaea isolated from Urmia Salt Lake, Iran, Halovarius luteus strain DA50T, was exposed to varying levels of simulated extraterrestrial conditions and compared to that of the bacteria Bacillus atrophaeus. Bacillus atrophaeus was selected for comparison due to its well-described resistance to extreme conditions and its ability to produce strong spore structures. Thin films were produced to investigate viability without the protective influence of cell multi-layers. Late exponential phase cultures of Hvr. luteus and B. atrophaeus were placed in brine and phosphate buffered saline media, respectively. The solutions were allowed to evaporate and cells were encapsulated and exposed to radiation, desiccation and vacuum conditions, and their post-exposure viability was studied by the Most Probable Number method. The protein profile using High Performance Liquid Chromatography and Matrix Assisted Laser Desorption/Ionization bench top reflector time-of-flight are explored after vacuum and UV-radiation exposure. Results showed that the change in viability of the spore-forming bacteria B. atrophaeus was only minor whereas Hvr. luteus demonstrated a range of viability under different conditions. At the peak radiation flux of 105 J/m2 under nitrogen flow and after two weeks of desiccation, Hvr. luteus demonstrated the greatest decrease in viability. This study further expands our understanding of the boundary conditions of astrobiologically relevant organisms in the harsh space environment.

Effects of ultraviolet-C on the spores of Bacillus subtilis and Bacillus velezensis suspension in phosphate buffered saline with their structural and molecular analysis using Raman-AFM imaging

Bacterial spores are of concern in food processing due to their ubiquity and resistance. This study seeks to determine the effect of ultraviolet C (UV-C) in the inactivation of spores of Bacillus subtilis and Bacillus velezensis that can result in enzymatic spoilage in foods using PBS as the suspension medium. Purified spore samples were treated under 1 pass in a UV-C reactor using 10 mL of spore inoculum with one dose of the radiation (410 mJ/cm2) for 10secs at room temperature. Aliquots of the treated samples were plated on tryptone soy agar supplemented with 0.6% glucose and the colonies counted. Flow cytometry analysis was done using 500 μL of both treated and control samples with a cell concentration of a ≥106 CFU/ml with propidium iodide (15 μM) and SYTO 9 (500 nM) used as live/dead stains. Samples were processed for microscopy (SEM and Raman-AFM Imaging). The maximum lethality is 2.5 for B. velezensis and the minimum is 0.1 for B. subtilis. Microscopic imaging of treated spores shows significant morphological disruption of the spore structure. The Raman spectroscopy analysis reveals the B. subtilis isolates to have the highest concentrations of dipicolnic acid (Ca+2DPA) as well as other compounds belonging to other functional groups. Flow cytometric analysis of treated spores reveals sub-populations unaccounted for by plate count. UV-C shows a promising application in the inactivation of resistant spores during processing of liquid foods such as milk.

DIAGNOSIS OF MYXOBOLUS BRAMAE (MYXOSPOREA: MYXOBOLIDAE) IN THE KIDNEYS TISSUE OF CARASOBARBUS LUTEUS AND HISTOPATHOLOGICAL CHANGES ASSOCIATED WITH INFECTION

This study was conducted during the period from March till the end of October 2018, to study the histopathological changes of Myxobolus bramae in kidney tissue of Carasobarbus luteus caught from Tigris River passing through Baghdad city. During the period of this study, a total of 60 fishes belonging to Carasobarbus luteus species from the family Cyprinidae were collected. The prevalence of infection with these protozoa was determinate (5.00%). Histopathological study due to M. bramae in the kidney tissue of C. luteus was done by using three types of stain: Hemotoxylin and eosin, giemsa and acid fast stain to observe plasmodia cyst and structures of spores. These changes characterized by tubular degeneration, necrosis, hyalinization of glomerular tuft, mild distension of Bowman᾿s space with a reduction in haemopoitic tissue together with inflammatory response, and accumulation of melanomacrophages at the site of infection. The results of this study revealed that Carasobarbus luteus from Tigris River at Baghdad city, it infected with Myxobolus bramae and this parasite cause severe histopathological changes in the infected kidneys tissue.

Learning from Nature: Bacterial Spores as a Target for Current Technologies in Medicine (Review).

The capability of some representatives of Clostridium spp. and Bacillus spp. genera to form spores in extreme external conditions long ago became a subject of medico-biological investigations. Bacterial spores represent dormant cellular forms of gram-positive bacteria possessing a high potential of stability and the capability to endure extreme conditions of their habitat. Owing to these properties, bacterial spores are recognized as the most stable systems on the planet, and spore-forming microorganisms became widely spread in various ecosystems.Spore-forming bacteria have been attracted increased interest for years due to their epidemiological danger. Bacterial spores may be in the quiescent state for dozens or hundreds of years but after they appear in the favorable conditions of a human or animal organism, they turn into vegetative forms causing an infectious process. The greatest threat among the pathogenic spore-forming bacteria is posed by the causative agents of anthrax (B. anthracis), food toxicoinfection (B. cereus), pseudomembranous colitis (C. difficile), botulism (C. botulinum), gas gangrene (C. perfringens).For the effective prevention of severe infectious diseases first of all it is necessary to study the molecular structure of bacterial spores and the biochemical mechanisms of sporulation and to develop innovative methods of detection and disinfection of dormant cells. There is another side of the problem: the necessity to investigate exo- and endospores from the standpoint of obtaining similar artificially synthesized models in order to use them in the latest medical technologies for the development of thermostable vaccines, delivery of biologically active substances to the tissues and intracellular structures. In recent years, bacterial spores have become an interesting object for the exploration from the point of view of a new paradigm of unicellular microbiology in order to study microbial heterogeneity by means of the modern analytical tools.

Proteome profile changes during poly-hydroxybutyrate intracellular mobilization in gram positive Bacillus cereus tsu1

BackgroundBacillus cereus is a bacterial species which grows efficiently on a wide range of carbon sources and accumulates biopolymer poly-hydroxybutyrate (PHB) up to 80% cell dry weight. PHB is an aliphatic polymer produced and stored intracellularly as a reservoir of carbon and energy, its mobilization is a key biological process for sporulation in Bacillus spp. Previously, B. cereus tsu1 was isolated and cultured on rapeseed cake substrate (RCS), with maximum of PHB accumulation reached within 12 h, and depleted after 48 h. Fore-spore and spore structure were observed after 24 h culture.ResultsQuantitative proteomic analysis of B. cereus tsu1 identified 2952 quantifiable proteins, and 244 significantly changed proteins (SCPs) in the 24 h:12 h pair of samples, and 325 SCPs in the 48 h:12 h pair of samples. Based on gene ontology classification analysis, biological processes enriched only in the 24 h:12 h SCPs include purine nucleotide metabolism, protein folding, metal ion homeostasis, response to stress, carboxylic acid catabolism, and cellular amino acid catabolism. The 48 h:12 h SCPs were enriched into processes including carbohydrate metabolism, protein metabolism, oxidative phosphorylation, and formation of translation ternary structure. A key enzyme for PHB metabolism, poly(R)-hydroxyalkanoic acid synthase (PhaC, KGT44865) accumulated significantly higher in 12 h-culture. Sporulation related proteins SigF and SpoEII were significantly higher in 24 h-samples. Enzymes for nitrate respiration and fermentation accumulated to the highest abundance level in 48 h-culture.ConclusionsChanges in proteome of B. cereus tsu1 during PHB intracellular mobilization were characterized in this study. The key enzyme PhaC for PHB synthesis increased significantly after 12 h-culture which supports the highest PHB accumulation at this time point. The protein abundance level of SpoIIE and SigF also increased, correlating with sporulation in 24 h-culture. Enzymes for nitrate respiration and fermentation were significantly induced in 48 h-culture which indicates the depletion of oxygen at this stage and carbon flow towards fermentative growth. Results from this study provide insights into proteome profile changes during PHB accumulation and reuse, which can be applied to achieve a higher PHB yield and to improve bacterial growth performance and stress resistance.

On the inactivation of Bacillus subtilis spores by surface streamer discharge in humid air caused by reactive species

The inactivation of Bacillus subtilis (ATCC 6633) spores deposited on a filter membrane was studied by using low-temperature plasma produced via surface dielectric barrier discharge. Spore samples were carefully prepared to avoid the formation of cell aggregates, and their inactivation was induced by multiple surface streamer discharge driven in a coplanar dielectric barrier discharge electrode geometry by an amplitude-modulated AC high voltage waveforms in humid air at atmospheric pressure. At a discharge duty cycle of 0.4, the surface dielectric barrier discharge is characterised by an average total power of 1.7 W (power density 1.5 W cm−2 and energy density ∼0.3 Wh l−1) and a low gas temperature of the plasma filaments of about 320 K. The spores were exposed by placing a sample holder at a fixed distance of 3 mm from the electrode surface covered by plasma filaments. Particular attention was paid to identifying sporicidal agents employed in the process of inactivation. Since treated samples did not come into direct contact with the streamer filaments and excessive heating was excluded thanks to the low energy density, our results indicate that the spores were inactivated mainly by reactive oxygen and nitrogen species such as O3, H2O2 and NO2–. Discharge-induced damage of the spore structure was evidenced via the detection of dipicolinic acid and leaking of intracellular components. We therefore conclude that B. subtilis spores were inactivated chemically, probably due to failure of the coat structure or membrane of the spore.

An attempt to create air sacs in spores? On the unusual spore structure in moss Encalypta longicollis

An attempt to create air sacs in spores? On the unusual spore structure in moss Encalypta longicollis

Bacillus subtilis builds structurally and functionally different spores in response to the temperature of growth.

Bacterial spores are commonly isolated from a variety of different environments, including extreme habitats. Although it is well established that such ubiquitous distribution reflects the spore resistance properties, it is not clear whether the growing conditions affect the spore structure and function. We used Bacillus subtilis spores of similar age but produced at 25, 37, or 42°C to compare their surface structures and functional properties. Spores produced at the 25°C were more hydrophobic while those produced at 42°C contained more dipicolinic acid, and were more resistant to heat or lysozyme treatments. Electron microscopy analysis showed that while 25°C spores had a coat with a compact outer coat, not tightly attached to the inner coat, 42°C spores had a granular, not compact outer coat, reminiscent of the coat produced at 37°C by mutant spores lacking the protein CotG. Indeed, CotH and a series of CotH-dependent coat proteins including CotG were more abundantly extracted from the coat of 25 or 37°C than 42°C spores. Our data indicated that CotH is a heat-labile protein with a major regulatory role on coat formation when sporulation occurs at low temperatures, suggesting that B. subtilis builds structurally and functionally different spores in response to the external conditions.

Expression and purification of recombinant puroindoline A protein in Escherichia coli and its antifungal effect against Aspergillus flavus.

Aspergillus flavus is the main cause of postharvest agricultural commodity loss. In this study, puroindoline A (PINA) protein was expressed in Escherichia coli, purified, and its antifungal properties against A. flavus were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the recombinant PINA protein was approximately 44 kDa. PINA exerted a powerful antifungal effect against A. flavus at 42.42 μg/mL on potato dextrose agar culture medium. Flow cytometry and scanning electron microscopy revealed that the spore morphology was damaged by PINA exposure; spores were depressed and broken, suggesting that the cell wall was impaired. Transmission electron microscopy and propidium iodide staining illustrated significant changes in intracellular spore structure, indicating cell membrane damage. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining indicated decreased mitochondrial membrane potential. Large nuclear condensation and DNA fragmentation were detected by 4',6-diamidino-2-phenylindole staining. The expression of genes related to the cell wall, cell membrane, and spore germination significantly changed in PINA-treated cells; this illustrated the probable mode of PINA action on A. flavus through cell wall destruction and triggered cell membrane, mitochondrial, and DNA damage leading to cell death. The antifungal mechanism of wheat PINA protein on A. flavus has been demonstrated in this study, and has potential application in preventing postharvest loss in the agricultural industry.

Morphological and Transcriptomic Analysis of the Inhibitory Effects of Lactobacillus plantarum on Aspergillus flavus Growth and Aflatoxin Production.

Lactobacillus plantarum, as a natural bio-preservative, has attracted a great deal of attention in recent years. In this study, 22 L. plantarum strains were tested against the aflatoxin-producing fungus, Aspergillus flavus; strain IAMU80070 showed the highest antifungal activity. At a concentration of 5 × 105 colony-forming units (CFU) mL−1, it completely inhibited A. flavus growth and decreased aflatoxin production by 93%. Furthermore, ultrastructural examination showed that IAMU80070 destroyed the cellular structure of hyphae and spores. To explore the inhibitory effect of IAMU80070 on A. flavus at the transcriptional level, transcriptome data were obtained and subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The aflatoxin biosynthetic process was the most significantly downregulated functional category, while genes implicated in the synthesis and organization of cell wall polysaccharides were upregulated. Quantitative real-time PCR results verified the credibility and reliability of the RNA sequencing data. These results provided insight into the transcriptome of A. flavus in response to the antagonistic effects of L. plantarum IAMU80070.

Analysis of the protein composition of the spindle pole body during sporulation in Ashbya gossypii.

The spores of fungi come in a wide variety of forms and sizes, highly adapted to the route of dispersal and to survival under specific environmental conditions. The ascomycete Ashbya gossypii produces needle shaped spores with a length of 30 μm and a diameter of 1 μm. Formation of these spores relies on actin and actin regulatory proteins and is, therefore, distinct from the minor role that actin plays for spore formation in Saccharomyces cerevisiae. Using in vivo FRET-measurements of proteins labeled with fluorescent proteins, we investigate how the formin AgBnr2, a protein that promotes actin polymerization, integrates into the structure of the spindle pole body during sporulation. We also investigate the role of the A. gossypii homologs to the S. cerevisiae meiotic outer plaque proteins Spo74, Mpc54 and Ady4 for sporulation in A. gossypii. We found highest FRET of AgBnr2 with AgSpo74. Further experiments indicated that AgSpo74 is a main factor for targeting AgBnr2 to the spindle pole body. In agreement with these results, the Agspo74 deletion mutant produces no detectable spores, whereas deletion of Agmpc54 only has an effect on spore length and deletion of Agady4 has no detectable sporulation phenotype. Based on this study and in relation to previous results we suggest a model where AgBnr2 resides within an analogous structure to the meiotic outer plaque of S. cerevisiae. There it promotes formation of actin cables important for shaping the needle shaped spore structure.

Revealing a new species of Ophioglossum (Ophioglossaceae-Pteridophyta) from India with palynological and phylogenetic implications

ABSTRACTIn recent years, Indian Ophioglossum L. taxonomy and systematic are receiving increasing interest from pteridologists as India has very rich germplasm of the genus. We present hereunder a new species of the genus Ophioglossum (Ophioglossaceae) from the marshy areas of southern region of Gujarat state, India. Ophioglossum hitkishorei sp. nov. can be diagnosed from other congeners by a combination of its smaller size, globose to subglobose, non-stoloniferous rhizome, number of trophophylls up to six, the absence of persistent leaf bases at top of rhizome and by very unique structure of spores. Spores are typically characterized by possessing granules deposition as exine strings bound around spores on the distal face and granulate assemblage on the proximal face of triradiate spores observed under SEM and also clearly distinct under light microscopic observations. As far as known, beaded strings of exine ornamentation have never been observed in any species of Ophioglossum in the world flora. Molecular study, based on two chloroplast DNA sequences trnL-F and psbA-trnH, also strongly supports O. hitkishorei sp. nov. as a distinct new species.

Surface morphology differences in Clostridium difficile spores, based on different strains and methods of purification

Infections linked to Clostridium difficile are a significant cause of suffering. In hospitals, the organism is primarily acquired through the faecal-oral route as spores excreted by infected patients contaminate the healthcare environment. We previously reported that members of the C. difficile group varied widely in their ability to adhere to stainless steel and proposed that these differences were a consequence of variations in spore architecture. In this study of clinical isolates and spore coat protein mutants of C. difficile we identified three distinct spore surfaces morphotypes; smooth, bag-like and “pineapple-like” using scanning electron microscopy (SEM). The frequency of each morphotype in a spore population derived from a single isolate varied depending on the host strain and the method used to produce and purify the spores. Our results suggest that the inclusion of a sonication step in the purification process had a marked effect on spore structure. In an attempt to link differences in spore appearance with key structural spore proteins we compared the morphology of spores of CD630 to those produced by CD630 variants lacking either CotE or BclA. While SEM images revealed no obvious structural differences between CD630 and its mutants we did observe significant differences (p < 0.001) in relative hydrophobicity suggesting that modifications had occurred but not at a level to be detectable by SEM.In conclusion, we observed significant variation in the spore morphology of clinical isolates of C. difficile due in part to the methods used to produce them. Sonication in particular can markedly change spore appearance and properties. The results of this study highlight the importance of adopting “standard” methods when attempting to compare results between studies and to understand the significance of their differences.

Arbuscular Mycorrhizae Morphotype-Based Identification Using Optical Microscopy

Structures of different AM taxa found in symbiosis with plants can stain from intense to weak after roots preparation for microscopy. Although arbuscules are distinctive features of AM, their overall shape can vary in association with two proliferation types: Arum and Paris. Auxiliary cells and vesicles are not omnipresent. Intraradical hyphae can present divergent types: straight/coiled, with “Y”/“H” connections. According to spore formation, there are five main AM morphotypes. Spore-based species identification takes into account: color, shape, size of spores and sporocarps as well as structures associated with them (cicatrix, pedicel, peridium, sporiferous saccule, sporogenous cell, subtending hypha). Description of subcellular structures of spores targets phenotypic and biochemical properties of layers using Melzer’s reagent and morphology of pre-germination structures when present.

Ozone disinfection of chlorine-resistant bacteria in drinking water

The wide application of chlorine disinfectant for drinking water treatment has led to the appearance of chlorine-resistant bacteria, which pose a severe threat to public health. This study was performed to explore the physiological-biochemical characteristics and environmental influence (pH, temperature, and turbidity) of seven strains of chlorine-resistant bacteria isolated from drinking water. Ozone disinfection was used to investigate the inactivation effect of bacteria and spores. The DNA concentration and cell surface structure variations of typical chlorine-resistant spores (Bacillus cereus spores) were also analysed by real-time qPCR, flow cytometry, and scanning electron microscopy to determine their inactivation mechanisms. The ozone resistance of bacteria (Aeromonas jandaei < Vogesella perlucida < Pelomonas < Bacillus cereus < Aeromonas sobria) was lower than that of spores (Bacillus alvei < Lysinibacillus fusiformis < Bacillus cereus) at an ozone concentration of 1.5 mg/L. More than 99.9% of Bacillus cereus spores were inactivated by increasing ozone concentration and treatment duration. Moreover, the DNA content of Bacillus cereus spores decreased sharply, but approximately 1/4 of the target genes remained. The spore structure exhibited shrinkage and folding after ozone treatment. Both cell structures and gene fragments were damaged by ozone disinfection. These results showed that ozone disinfection is a promising method for inactivating chlorine-resistant bacteria and spores in drinking water.

An Efficient Adaptive Algorithm for Electron Microscopic Image Enhancement and Feature Extraction

In this article, a block-based adaptive contrast enhancement algorithm has been proposed, which uses a modified sigmoid function for the enhancement and features extraction of electron microscopic images. The algorithm is based on a modified sigmoid function that adapts according to the input microscopic image statistics. For feature extraction, the contrast of the image is very important and authentic property by which this article enhances the visual quality of the image. In this work, for better contrast enhancement of image, a block based on input value, combined with a modified sigmoid function that is used as contrast enhancer provides better EMF values for a smaller block size. It provides localized contrast enhancement effects adaptively which is not possible using other existing techniques. Simulation and experimental results demonstrate that the proposed technique gives better results compared to other existing techniques when applied to electron microscopic images. After the enhancement of microscopic images of actinomycetes, various important features are shown, like coil or spiral, long filament, spore and rod shape structures. The proposed algorithm works efficiently for different dark and bright microscopic images.

A system for the expression and release of heterologous proteins from the core ofBacillus subtilisspores

Methodologies that exploit the durability of Bacillus subtilis spores by displaying heterologous proteins or antigenic molecules on the spore surface for mucosal vaccine delivery and other applications are well established. Here we extend the concept by engineering spores intended as oral delivery vehicles for therapeutic proteins. The method is exemplified by the expression and deposition of human growth hormone in the developing spore core, where the protein is shielded from physicochemical and biological degradation by the protective spore structure. Lysates from physically disrupted spores are shown to stimulate differentiation of a pre-adipocyte cell line to mature adipocyte cells, indicating that the spore-core located human growth hormone is folded correctly and functional. We also introduce a methodology for controlled release of heterologous proteins from the spore core, which utilises components of the PBSX prophage to lyse spores during germination and outgrowth. With further development, spore core expression, coupled with an engineered autolytic germination mechanism, may permit the use of spores as oral delivery carriers of therapeutic proteins.

Enhanced method for High Spatial Resolution surface imaging and analysis of fungal spores using Scanning Electron Microscopy

Efficient, fast and new micro-analytical methods for characterization of ultrastructures of fungal spores with electron microscopy are very much required and essential. SEM analysis of biological materials, especially fungi, requires optimal preparation of the specimen and often requires the usage of dried samples which demands a challenging sample preparation. In the present investigation, we described a fast and improved method for the preparation of fungal specimen for scanning electron microscopy (SEM). The fungus, Curvularia lunata was grown on the surface of sterile Whatman No.1 filter paper which was overlaid on Potato Dextrose Agar (PDA) medium, gold coated immediately after removal from the growth medium and subjected to imaging. Generally, SEM imaging is done with samples that were fixed with chemical fixatives, dehydrated and gold coated specimens, but here we describe an easy and more efficient sample preparation for SEM which enabled enhanced image quality and precision visualization of fungal cells, especially the spores. The developed method has enabled the analysis of even the robust samples like fungal spores that to eliminating special temperature requirement. The ultimate goal was to develop an improved protocol/method applied to analysis of fungal spores with greater coverage about fungal specimen preparation. This method permits the use of rapid sample preparation and will allow us to imaging of individual spore or conidia structures in the context of fungal cell architecture which clarifies our understanding in fungal taxonomy/biology.