Free Spores Research Articles

Advanced Nosema bombycis Spore Identification: Single-Cell Raman Spectroscopy Combined with Self-Attention Mechanism-Guided Deep Learning.

Nosema bombycis (Nb) has been considered a dangerous pathogen, which can spread rapidly through free spores. Nowadays, pebrine disease caused by Nb spores is a serious threat to silkworms, causing huge economic losses in both the silk industry and agriculture every year. Thus, how to accurately identify living Nb spores at a single-cell level is greatly demanded. In this work, we proposed a novel approach to accurately and conveniently identify Nb spores using single-cell Raman spectroscopy and a self-attention mechanism (SAM)-guided convolutional neural network (CNN) framework. With the assistance of SAM and data augmentation methods, an optimal CNN model can not only efficiently extract spectral feature information but also construct potential relationships of global spectral features. Compared with the case without both SAM and data augmentation, the average prediction accuracy of Nb spores from nine different Bombyx mori larvae can be significantly developed by almost 18%, from original 83.93 ± 4.88% to 99.27 ± 0.25%. To visualize the individual classification weight, a local feature extraction strategy named blocking individual Raman bands was proposed. According to the relative weight, these four Raman bands located at 1658, 1458, 1127, and 849 cm-1, mainly contribute to the high prediction accuracy of 99.27 ± 0.25%. It is worth noting that these Raman bands were also highlighted by the weight curve of SAM, indicating that the four Raman bands proposed by our optimal CNN model are reliable. Our findings clearly show that single-cell Raman spectroscopy combined with SAM-mediated CNN configuration has great potential in performing early diagnosis of Nb spores and monitoring pebrine disease.

Myxococcus xanthus fruiting body morphology is important for spore recovery after exposure to environmental stress.

Environmental microorganisms have evolved a variety of strategies to survive fluctuations in environmental conditions, including the production of biofilms and differentiation into spores. Myxococcus xanthus are ubiquitous soil bacteria that produce starvation-induced multicellular fruiting bodies filled with environmentally resistant spores (a specialized biofilm). Isolated spores have been shown to be more resistant than vegetative cells to heat, ultraviolet radiation, and desiccation. The evolutionary advantage of producing spores inside fruiting bodies is not clear. Here, we examine a hypothesis that the fruiting body provides additional protection from environmental insults. We developed a high-throughput method to compare the recovery (outgrowth) of distinct cell types (vegetative cells, free spores, and spores within intact fruiting bodies) after exposure to ultraviolet radiation or desiccation. Our data indicate that haystack-shaped fruiting bodies protect spores from extended UV radiation but do not provide additional protection from desiccation. Perturbation of fruiting body morphology strongly impedes recovery from both UV exposure and desiccation. These results hint that the distinctive fruiting bodies produced by different myxobacterial species may have evolved to optimize their persistence in distinct ecological niches.IMPORTANCEEnvironmental microorganisms play an important role in the production of greenhouse gases that contribute to changing climate conditions. It is imperative to understand how changing climate conditions feedback to influence environmental microbial communities. The myxobacteria are environmentally ubiquitous social bacteria that influence the local microbial community composition. Defining how these bacteria are affected by environmental insults is a necessary component of predicting climatic feedback effects. When starved, myxobacteria produce multicellular fruiting bodies filled with spores. As spores are resistant to a variety of environmental insults, the evolutionary advantage of building a fruiting body is not clear. Using the model myxobacterium, Myxococcus xanthus, we demonstrate that the tall, haystack-shaped fruiting body morphology enables significantly more resistance to UV exposure than the free spores. In contrast, fruiting bodies are slightly detrimental to recovery from extended desiccation, an effect that is strongly exaggerated if fruiting body morphology is perturbed. These results suggest that the variety of fruiting body morphologies observed in the myxobacteria may dictate their relative resistance to changing climate conditions.

Ceratomyxa matosi n. sp. (Myxozoa: Ceratomyxidae) parasitizing the gallbladder of Boulengerella cuvieri (Characiformes: Ctenoluciidae) State of Amapá, Brazilian Amazon.

Myxozoa is a class of the Phylum Cnidaria made up of endoparasites from aquatic habitats. The genus Ceratomyxa preferentially infects marine fish, with the gallbladder being the main site parasitized. This study aimed to describe a new species of Ceratomyxa found in this organ in Boulengerella cuvieri using morphological, morphometric characterization and phylogenetic analysis of 18S rDNA gene sequences. Specimens of B. cuvieri were collected, anesthetized, desensitized and biometric measurements were performed. The organs were analyzed under a stereomicroscope and fragments of internal organs were extracted for light microscopy analysis, preserved in 80% ethanol for 18S rDNA gene analysis and fixed in Davidson solution for histological processing. Free spores of Ceratomyxa were observed in the gallbladder, in plasmodia with wave-like movements, with the following dimensions: spore width (24.5 ± 0.4) µm, spore length (5.2 ± 0.3) µm, polar capsule width (1.8 ± 0.2) µm, polar capsule length (2.1 ± 0.3) µm, number of polar tubule turns (4-5) and 100% prevalence. Phylogenetic analysis confirmed that Ceratomyxa matosi n. sp. is a new species, grouped with other freshwater Ceratomyxa species from the Amazon, representing the second description of species of this genus in the state of Amapá.

Spore Abundance and Morphology of Arbuscular Mycorrhizal Fungal under Conservation Agriculture

Soil is full of microbial diversity and microbes present in soil interact diversely with other living form present in soil e.g., soil microbes and plants. These interactions range from symbiosis to parasitic. Among these, the most predominating mutualistic symbiotic relation is mycorrhiza (associated with ∼80% of terrestrial plant species), formed between arbuscular mycorrhizal (AM) fungi and vascular plants. An experiment was conducted under three cropping system rice-maize-cowpea (RMCp), rice-wheat-green gram (RWGg) and rice-cauliflower-bororice cropping (RCfBr) to study influence of three different tillage intensities i.e., CT (conventional tillage), RT (reduced tillage), and ZT (zero tillage) with five different levels of fertilizer and residue application, R1 (0%Residue & 100%RDF), R2(100%Residue & 50%RDF), R3 (100%Residue & 75%RDF), R4 (50%Residue & 100%RDF), R5 (50%Residue & 75%RDF), on AMF spore load and diversity.The experimental design followed was split plot with tillage was main plot treatment and residue as sub plot treatment. Result showed that AMF spores and diversity are significantly influenced of degree of soil disturbance under different tillage practices and cropping system in practice, however residue application imparted non-significant influence on AMF spore load and diversity. A significant lower spore count was observed under CT as degree of soil disturbance is excessive high under CT compared to RT and ZT. With respect to spore diversity, round (shape), brown (color) and free spores were predominating spore type. In reference to spore shape abundance sequence was round>spherical> oval while for spore color it was brown>yellow>hyaline and >90 % spore was free while attached spores were <5%. In case of AMF spore count influence of cropping system was more prominent compared to other factors (i.e., tillage and residue). In RMCp and RWGg, significant increase inspore count was observed compared to RCfBr cropping system. On second year of experimentation, in RCfBr cropping system a significant decline in spore number was detected due to inclusion of cauliflower which act as non-host of AMF. Hence to attain benefits associated with AMF to arable crop we must reduce level of soil disturbance and avoid crucifer crops in cropping system.

Morphological and physiological-biochemical variability of spore-forming bacteria isolated from the agrocoenosis of winter wheat

From an agrocoenosis of winter wheat (Triticum aestivum L.; phylloplane and rhizosphere of the root system; typical chernozem, soil column measuring up to 40 cm), using the classical microbiological methods, we had isolated soil bacteria and characterized them according to the morphological features as representatives of Gram-positive and spore-forming bacteria of Bacillus sp. genus. In the earing-swelling phase of grain, the screening studies found non-pigmented forms of colonies of bacterial isolates, 19 of which were classified to colonial-morphological diversity of R-type with the diameter of 7 to 13 mm. The analysis of physiological condition of cells of populations of soil isolates revealed technologic specificity according to parameters of spore formation in different conditions and incubation time (up to 48–72 h). We observed 90.0% of free spores in axenic cultures as early as after 72 h of cultivation and no more than 10.0% of prospores in the studied monoisolates with stable morphologic traits. Isolates Н10 and Н45 demonstrated the ability to grow in higher cultivation temperatures (+37…+40 °С). According to environmental рН, isolates were able to grow in рН ranging 4.5–8.0. Differential diagnostic testing revealed that as the source of carbon, with formation of acid, soil isolates used arabinose, xylose, mannitol, glucose, galactose, fructose, maltose, sorbitol, glycerin, dextrin, starch, rhamnose and dulcite (with development of alkaline). There was observed active use of mineral forms of nitrogen: ammonium salt and nitrates, aminoacids and proteins. The isolates hydrolyzed casein, gelatin, starch, and litmus was being reduced in the young during growth in milk with litmus. They also exerted catalase activity and were oxidase-positive. Biochemical testing using API test system determined that the studied isolated bacteria differed by a range of fermentation carbohydrates, reduction of nitrates. In the conditions of submerged fermentation, isolates Н38 and Н40 grew in heightened temperature ranges of cultivation (40 °С) for 48 h (according to fact of spore development). Therefore, according to the key morphologic and biochemical traits, strains Н3, Н10, Н13, Н36, Н38, Н40, Н43, Н45 were similar to such of reference strain B. subtilis 8A, and were identified to Bacillus sp., species B. subtilis.

Morphological and Phylogenetic Features of Ceratomyxa macapaensis n. sp. (Myxozoa: Ceratomyxidae) in Mesonauta festivus Heckel, 1840 (Cichliformes: Cichlidae) from the Eastern Amazon Region.

The class Myxozoa consists of microscopic spores that typically present uniform morphological simplifications, containing one or two sporoplasms surrounded by valve cells. The present study analysed the morphological and phylogenetic characteristics of what proved to be a new species of Ceratomyxa found in the gallbladder of Mesonauta festivus Heckel, 1840 captured on the Piririm River in the municipality of Macapá, in Amapá state, Brazil. The fish were collected in gillnets, and were transported alive to Amapá State University in Macapá for the analysis of the organs. Crescent- or arch-shaped free spores were observed under light microscopy, containing two polar capsules and a suture line, which is typical of the morphology of the genus Ceratomyxa, contained within plasmodia that had snake-like movements. The gallbladder was conserved in 80% alcohol for the analysis of the 18S rDNA gene, and in Davidson solution for standardhistology. Ceratomyxa spores (n = 20) were observed in 11 of the 50 samplesanalysed, and thus had a prevalence of 22%, and had a mean length of 4.2 ± 0.5µm, mean thickness of 22.75 ± 0.3µm, and two polar capsules, 1.86 ± 0.3µm long and 1.63 ± 0.1µm thick. The phylogenetic analysis indicated that the new species forms a cluster with other freshwater Amazonian Ceratomyxa species, described previously. The new species represents the first description of a taxon of the genus Ceratomyxain fish from the Brazilian state of Amapá.

Ultrastructural and pathogenicity of Brevibacillus laterosporus against sinantropic muscoid dipterans.

Brevibacillus laterosporus has entomopathogenic potential against several orders of insects and its wide bioactivity is associated with a variety of strain-specific molecules. In order to avoid the use of synthetic insecticides, along with the need to control insect pests, microbial control has been widely used. Muscoid dipterans are known for their medical-veterinary and sanitary importance, and synanthropy. The enormous biotechnological potential of B. laterosporus has been demonstrated, but there are still few studies with muscoid dipterans. The aim of the study was to verify the mortality of B. laterosporus NRS590 on synanthropic flies and to characterize its different cell stages ultrastructurally. The flies were collected from garbage bins and the colonies were adapted to the laboratory conditions. Bioassays with neo larvae were carried out from the bacterial growth in the phases: vegetative (6hr), sporangium (20 hr), and free spores (44 hr). An aliquot of each phase was collected for Transmission (TEM) and Scanning Electron Microscopy (SEM). The effectiveness of NRS590 was observed in the sporulation phase, where the corrected mortality was 83.3, 85.1, and 99% for Chrysomya megacephala, Chrysomya putoria, and Musca domestica, respectively. The parasporal body was observed in detail on the entire spore surface. Although our knowledge of this bacterium is growing, it remains to be determined the real virulence factors responsible for the wide entomopathogenic activity observed on muscoid dipterans. Therefore, this study can provide subsidies for the improvement of efficient and safe microbial control techniques for the environment and living beings.

Ellipsomyxa tucujuensis n. sp. (Myxozoa: Ceratomyxidae), a parasite of Satanoperca jurupari (Osteichthyes: Cichlidae) from the Brazilian Amazon.

The present study describes a new coelozoic, eukaryotic microparasite of the genus Ellipsomyxa Køie, 2003 (Ceratomyxidae: Myxozoa) found parasitizing the gallbladder of Satanoperca jurupari Heckel, 1840 collected in the Curiaú River Environmental Protection Area in Macapá, Amapá state, Brazil. The fish were collected using mesh cast net. The gallbladders were examined, preserved in 80% alcohol for molecular analysis (SSU rDNA gene), and fixed in Davidson for histological slide preparation. The new parasite had a prevalence of 81% in the gallbladder, asymmetric plasmodia, irregular free spores in the bladder fluid, with no cyst formation. The spores are elliptical, with characteristics of the genus Ellipsomyxa, and they had a mean length of 10.11 (8.56-10.5) μm, mean width of 7.81 (5.96-9.56) μm, and thick walls. The polar capsules are sub-spherical in shape, slightly asymmetrical, with a mean length of 3.12 (2.31-3.99) μm and mean width of 2.5 (2.22-2.95) μm, containing polar filament with five or six coils perpendicular to the longitudinal axis of the capsule. The Bayesian Inference assigned the new species to a subclade formed by a lineage of Ellipsomyxa species from the Amazon region. Ellipsomyxa tucujuensis n. sp. is the sixth species of this genus described in fish from the Amazon region, and the first for the state of Amapá.

Effect of Batrachochytrium Dendrobatidis on Anura Populations Batrachochytrium

Chytrium (Batrachochytrium Dendrobatidis) is a chytrium fungus that causes amphibian chytriomycosis. They were first discovered in 1998 and have been responsible for a large number of amphibian deaths over the next decade. Chytrid fungus grows on amphibian skin and produces free aquatic spores. They are widely distributed in deserts and lowland forests to cold hilltops. They can cause disease in upland (low temperature) amphibians, causing significant mortality. Chytrid infection has been associated with large numbers of amphibian deaths in north, south and central America, Europe and Australia. In this paper, the author summarized several factors influencing the transmission of chytrid by analyzing the existing data and data, including the transmission route, the worldwide distribution range and harm degree of chytrid, and the existing research and discussion on the elimination and prevention methods of chytrid. After collecting and analyzing data and literature, the author comes to the following conclusions: Firstly, the fungus originated in Africa and spread around the world through trade in animals such as xenopus africanus, and was endemic in Africa for many years before spreading around the world. Secondly, chytridiomycosis is one of the important factors contributing to the extinction of some amphibians in many places, including Africa and Australia. Thirdly, the factors that will affect the chytrid strain of chytrid fungus, the active degree and spread range of factors are temperature, human activities, amphibian’s own immunity, chemicals in pesticides, etc.

Enhancement of the Anti-inflammatory Effect of Bromelain by Its Immobilization on Probiotic Spore of Bacillus cereus.

The therapeutic application of bromelain is limited due to its sensitivity to operating conditions such as high acidity, gastric proteases in the stomach juice, chemicals, organic solvents and elevated temperature. We hypothesized that bromelain immobilized on probiotic bacterial spores would show enhanced therapeutic activity through possible synergistic or additive effects. In this study, the oedema inhibition potential of bromelain immobilized on probiotic Bacillus spores was compared to the free enzyme using the carrageenan paw oedema model with Wistar rats. In batch A rats (carrageenan-induced inflammation 30min after receiving oral treatments), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed the highest oedema inhibition, 89.20±15.30%, while group 4 treated with a lower dose of free bromelain had oedema inhibition of 60.25±13.00%. For batch B rats (carrageenan-induced inflammation after receiving oral treatment for three days), group 7 rats treated with a lower dose of spore-immobilized bromelain suspension showed higher inhibition percentage (81.94±8.86) than group 4 treated with a lower dose of free bromelain (78.45±4.46) after 24h. Our results showed that used alone, the enzyme and the spores produced oedema inhibition and improved the motility of the rats. The spore-immobilized bromelain formulation performed approximately 0.9-fold better than the free bromelain and the free spores at the lower evaluated dose.

Co-immobilized spore laccase/TiO2 nanoparticles in the alginate beads enhance dye removal by two-step decolorization.

Combinatorial application of different dye removal methods with specific features can lead to a novel and robust decolorizing system. In this study the bacterial spore laccase and TiO2 nanoparticles were co-entrapped to enhance dye degradation. The optimum entrapment conditions were achieved in the presence of alginate 2% (w/v) and Ca2+ (0.2M), Cu2+ (0.05M) and Zn2+ (0.25M) as matric polymer and counterions, respectively. Immobilized laccase showed a wide range of pH and temperature stability in comparison to the free spores. The entrapped degradation systems include single laccase, single TiO2, laccase + TiO2 (one-step remediation), TiO2/laccase (two-step remediation), and laccase/TiO2 (two-step remediation) that result to the 22%, 26% 45.6%, 47.6%, and 69.3% indigo carmine decolorization in 60 min. In the kinetic studies, the half-life of indigo carmine (25 mg/l) in the remediation processes containing laccase, TiO2, laccase + TiO2, TiO2/laccase, and laccase/TiO2 was calculated as 173, 138, 161, 115, and 57 min, respectively. The degradation products by co-entrapped system were not toxic against Sorghum vulgare. The results showed two-step decolorization by co-entrapped spore laccase and TiO2 nanoparticles, including the pretreatment of dye by laccase, and then, treatment by TiO2 has potential for degradation of indigo carmine.

Impact of a Gastrointestinal Stable Probiotic Supplement Bacillus coagulans LBSC on Human Gut Microbiome Modulation

Bacillus coagulans LBSC showed stability in acidic pH, bile and simulated human gastrointenstinal juices. Under static gut model, when passed through oral, gastric and intestinal phases, B. coagulans LBSC was found to be stable as free viable spores and also with various foods such as milk and baby foods, as well as American and European diets. In human studies, modulation of gut microbiota by B. coagulans LBSC was comprehended by whole genome metagenome analysis of fecal samples obtained from pre- and post-treatment of irritable bowel syndrome (IBS) patients. B. coagulans LBSC treatment showed positive modulation in gut microbiota, especially up regulation of phyla such as Actinobacteria and Firmicutes, whereas down regulation of Bacteroids, Proteobacteria, Streptophyta and Verrucomicrobia. Simultaneously, it has altered various microbiota associated metabolic pathways to create the normalcy of gut microenvironment.

Protist Interactions and Community Structure During Early Autumn in the Kerguelen Region (Southern Ocean)

This study investigated protist community composition and biotic interactions focusing on microplankton at four distinct sites around the Kerguelen Islands (Southern Ocean) after the summer phytoplankton bloom. Protist diversity in different size fractions, sampled with Niskin bottles and plankton nets, was assessed by sequencing of the V4 18S rDNA region. Combining different approaches, i.e. sequencing of different plankton size fractions, and isolation and sequencing of single cells, provided new insights into microbial interactions in protist communities. The communities displayed high variability, including short-term fluctuations in relative abundance of large protists (>35μm) highlighted by the plankton net samples. Size fractionation of protist communities showed high concentrations of free Syndiniales spores but relatively few Syndiniales associated with microplankton, suggesting low parasitic infection in early autumn. Co-variance network analyses and sequencing of individually isolated single cells highlighted the important role of Rhizaria as consumers of a wide range of different diatom taxa. The data also raised the hypothesis that different Syndiniales clades might be directly or indirectly associated with some diatom genera, thus suggesting a potentially wider host range of these parasites than has been previously reported. These associations and the potential impact on carbon fluxes are discussed.

Evaluation and Microanalysis of Parasitic and Bacterial Agents of Egyptian Fresh Sushi, Salmo salar.

The present study aimed to evaluate the quality of fresh sushi in Egypt. Fifty samples of sushi (Salmo salar) were collected from restaurants in Alexandria, Egypt. Paraffin, semi-thin and ultra-thin sections were used for parasitological analysis by light and transmission electron microscopy. Bacteria were isolated by the dilution plate and direct plate methods and identified by a Vitek system. Twenty (40%) of the total examined samples showed microsporidia and helminth metacercariae infections. Histochemical stains showed distinct pinkish-red pyriform microspores embedded in muscular tissue stained with Gram, periodic acid-Schiff (PAS), and Ziehl-Neelsen (ZN) stains. Semi-thin sections showed double membrane xenoma-inducing granulomas containing spores at different developmental stages. Empty sporophorous vesicles and free spores were observed in the electron microscopic images. A bacteriological assay showed forty samples (80%) contaminated with human pathogenic bacteria with the average total bacterial counts ranging from 32 to 526 CFU/g. Four species of human pathogenic bacteria were identified in the examined samples, namely Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Serratia plymuthica in 40, 38, 11, and 6 samples, respectively. These constitute the first record of fresh sushi product in Egypt and indicate the potential pathogenicity associated with raw seafood products.

New data on Myxobolus imparfinis (Cnidaria, Myxosporea): host, distribution, and ultrastructural morphology.

A mass of free myxozoan spores was found in the gill filaments of specimens of Cetopsorhamdia iheringi Schubart and Gomes, 1959, popularly known as "three-barbeled catfishes" (Heptapteridae, Siluriformes) collected in streams of the Middle Paranapanema River, Upper Paraná River basin, in the state of São Paulo, Brazil. Morphological and molecular analysis identified the spores as Myxobolus imparfinis Vieira, Tagliavini, Abdallah and Azevedo, 2018. The ultrastructural morphology of this parasite is described here for the first time. Differences were observed in the number of coils of the polar filament as well as some organelles not previously described for this species. Asynchronous development was also observed, with the presence of both mature and immature spores. This is the first report of a myxozoan parasitizing C. iheringi and the first geographical record of myxozoan parasites in streams of the Middle Paranapanema River. The new data improve the original description of the species and add to the knowledge of host-parasite interactions and distribution.

Skin nodules associated with parasitism with Henneguya sp. (Cnidaria: Myxosporea) in the neotropical fish Cyphocharax modestus

A new myxozoan species, Henneguya sp., is described based on material from skin of Cyphocharax modestus. Mature myxospores are were elongate and ellipsoidal, measuring 21.4 ± 1.2 (19.4–23.2) μm in total length, 5.1 ± 0.3 (4.5–5.8) μm in width, 11.9 ± 0.5 (10.9–12.7) μm in body length and 9.6 ± 0.7 (8.4–10.5) μm in length of the caudal process. The polar capsules were elongated and had unequal sizes, with length of 5.1 ± 0.4 (4.5–6.0) μm and 5.6 ± 0.4 (4.9–6.3) μm for smaller and larger respectively and width of 1.8 ± 0.2 (1.4–2.0) μm. The larger polar capsule had 8 turns in polar filament while the smaller polar capsule had 5 turns in polar filament. The macroscopic analysis revealed the presence of large nodules, which were located before and after the dorsal fin of the hosts. The histopathological analysis showed the development of nodules filled with plasmodia, surrounded by loose connective tissue, developed in the dermis of the skin. Many cysts containing countless spores, as well as free spores, were located in the dermis and hypodermis of the hosts, causing the disorganization of the connective tissue that is responsible for the support. This is the first record of a Henneguya species in C. modestus.

Evaluation of the reproductive potential of Encephalitozoon intestinalis in four different cell line

Microsporidia are parasites that can cause infections in many vertebrate and invertebrate organisms and produce small spores resistant to environmental conditions. As they are obligate intracellular parasites, axenic cultures cannot be performed. The aim of this study was to investigate the reproductive potential of the parasite in human colon epidermal adenocarcinoma (Caco-2), human monocytic (U937), African green monkey renal epithelial (VERO) and human kidney epithelial (HEK-293) cell lines of tissue and organs where the parasite is located by following the culture of the parasites and the amount of spores for six weeks. RPMI-1640 medium was used for the cultivation of U937 cells, while DMEM was used for other cell lines and the immature U937 cells were stimulated with Phorbol-12-Myristate-13-Acetate before infection. All of the host cell groups were infected with freshly collected Encephalitozoon intestinalis spores in ratio 1:30 and free spores in the culture media were removed after overnight incubation at 37°C under 5% CO2 condition for parasite invasion. The first release of the spores from the infected cells was observed and recorded by following for six weeks. Furthermore, the spore density released from each cell groups was evaluated by measuring the parasite load by Thoma cell counting chamber and quantified by real-time PCR. As a result of the study, it was observed that four cell lines could be infected by E.intestinalis and the spore production can be maintained for six weeks. It was observed that the monolayer macrophages and CaCo-2 cells, started to be detached from the culture flasks in few days following the parasite invasion, thus decreasing the number of host cells. After 1-2 weeks, HEK-293 cells were also detached from the surface, thus negatively affected the pure spore production by contaminating the media with dead host cell suspension. Spores started to appear in VERO cell media at the end of the second week after initial infection, while it took longer time for other cells to start releasing spores. Over the course of six weeks, the VERO cell line had the highest spore-producing potential among the other cell lines. In conclusion, this study compared the potential for reproduction of E.intestinalis in three human cell lines and monkey originated VERO cell line. This study demonstrated that cells derived from the tissues or organs where Microsporidia species causes disseminated infections could be infected by the parasitic spores in vitro. Additionally, the parasite can survive and propagate longer than six weeks. The authors believe that the results of this study will contribute to the further studies related to the parasite in the area of genetics, pharmacology, biochemistry, immunology and eradication studies.

Bacteria, mould and yeast spore inactivation studies by scanning electron microscope observations

Spores are the most resistant form of microbial cells, thus difficult to inactivate. The pathogenic or food spoilage effects of certain spore-forming microorganisms have been the primary basis of sterilization and pasteurization processes. Thermal sterilization is the most common method to inactivate spores present on medical equipment and foods. High pressure processing (HPP) is an emerging and commercial non-thermal food pasteurization technique. Although previous studies demonstrated the effectiveness of thermal and non-thermal spore inactivation, the in-depth mechanisms of spore inactivation are as yet unclear. Live and dead forms of two food spoilage bacteria, a mould and a yeast were examined using scanning electron microscopy before and after the inactivation treatment. Alicyclobacillus acidoterrestris and Geobacillus stearothermophilus bacteria are indicators of acidic foods pasteurization and sterilization processes, respectively. Neosartorya fischeri is a phyto-pathogenic mould attacking fruits. Saccharomyces cerevisiae is a yeast with various applications for winemaking, brewing, baking and the production of biofuel from crops (e.g. sugar cane). Spores of the four microbial species were thermally inactivated. Spores of S. cerevisiae were observed in the ascus and free form after thermal and HPP treatments.Different forms of damage and cell destruction were observed for each microbial spore. Thermal treatment inactivated bacterial spores of A. acidoterrestris and G. stearothermophilus by attacking the inner core of the spore. The heat first altered the membrane permeability allowing the release of intracellular components. Subsequently, hydration of spores, physicochemical modifications of proteins, flattening and formation of indentations occurred, with subsequent spore death. Regarding N. fischeri, thermal inactivation caused cell destruction and leakage of intracellular components. Both thermal and HPP treatments of S. cerevisiae free spores attacked the inner membrane, altering its permeability, and allowing in final stages the transfer of intracellular components to the outside. The spore destruction caused by thermal treatment was more severe than HPP, as HPP had less effect on the spore core. All injured spores have undergone irreversible volume and shape changes. While some of the leakage of spore contents is visible around the deformed but fully shaped spore, other spores exhibited large indentations and were completely deformed, apparently without any contents inside. This current study contributed to the understanding of spore inactivation by thermal and non-thermal processes.

Removal of Cr(VI) from solution by free and immobilised spores of Aspergillus niger

Despite the current interest in microbial methods for the detoxification of effluents, relatively little work has focused on the characterisation of metal uptake by small Aspergillus niger spores, particularly for heavy metals present at different and low concentrations. The objective of this study was to determine the ability of A. niger spores (free spores and as immobilised biosorbent) to remove toxic metals from an industrial wastewater as a batch system. The simultaneous influences of the mass of pH and biomass on the ability of spores to uptake of Cr(VI) were analysed. The free and immobilised biosorbent removed Cr(VI) efficiently with a maximum metal removal capacity of 55 and 40 mg/g, respectively. The biosorption mechanism was also investigated using Fourier transfer infrared and scanning electron microscopy. Immobilised spores of A. niger were more stable in solutions at different pH levels and allowed easier separation from aqueous solution than free spores, suggesting significant potential for future industrial application.

Spore Analysis and Tetrad Dissection of Schizosaccharomyces pombe.

Here we describe the processing of Schizosaccharomyces pombe spores in batches (random spore analysis) or through tetrad dissections. Spores are usually prepared from matings between haploid strains (producing zygotic asci) or from sporulating diploids (producing azygotic asci). In random spore analysis, a snail enzyme preparation is used to digest the walls of asci to release free spores that are diluted and plated to form colonies. In tetrad dissection, a needle attached to a micromanipulator is used to pick asci and separate spores. Tetrad dissection has traditionally been the method of choice for genetic mapping and is very useful in the study of genetic interactions (e.g., suppressor analysis). It is also the preferred method for routine crosses because it ensures that every colony stems from a single spore. This can never be certain in random spore analysis.