scholarly journals Strategy for the Removal of Satellite Bacteria from the Cultivated Diatom

Diversity ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 382
Author(s):  
Yulia Zakharova ◽  
Artem Marchenkov ◽  
Nadezhda Volokitina ◽  
Aleksey Morozov ◽  
Yelena Likhoshway ◽  
...  
Keyword(s):  
Extracellular Polymeric Substances ◽  
High Throughput Sequencing ◽  
Single Cells ◽  
Axenic Culture ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Bacterial Cells ◽  
Genetic Studies ◽  
Bacterial Dna ◽  
Total Dna

Multiple ecological and genetic studies of diatom algae require an axenic culture. However, algae-associated bacterial biofilms often form in diatom-produced mucus, both during creation of monoclonal cultures from single cells and during biomass growth, and they may be difficult to remove. In this work, we describe a protocol for removing associated bacteria from a monoclonal culture of Ulnaria danica isolated from Lake Baikal. The axenization strategy involves selecting the latent phase of diatom growth, multiple washes to remove extracellular polymeric substances and bacterial cells, filter deposition, and treatment with antibiotics that are not toxic for diatoms. The absence of bacteria during these stages was controlled by light microscopy with Alcian blue staining for mucus, epifluorescent microscopy with DAPI (4′,6-diamino-2-phenylindole) staining for bacterial DNA, and scanning electron microscopy of the diatom cell surface. High-throughput sequencing of a 16S rRNA fragment, amplified with universal bacterial primers, from total DNA of a final culture failed to detect any bacterial contamination, confirming successful axenization. A detailed comparative description of all stages of our protocol may prove useful in developing axenic cultures of other diatoms for various ecological and genetic studies.

scholarly journals THU0058 ENHANCEMENT OF CARTILAGE REGENERATION EFFICIENCY WITH HUMAN ADIPOSE STEM CELL THREE-DIMENSIONAL SPHEROID

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 241.2-241
Author(s):  
J. Y. Ko ◽  
E. Lee ◽  
J. Kim ◽  
G. I. Im
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Single Cells ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Cartilage Regeneration ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Culture Dish

Background:3D (three-dimensional) cell culture technology has been researched steadily because of its high potential of biocompatibility compared to single cells since 1990s, and is being developed to 3D spheroids recently. Spheroids are considered to reflect the natural organization of cells better than 2D cell cultures, and stem cells spheroids have been studied extensively in therapeutic transplantation. Stem cells were considered as a method of replacing autologous chondrocyte in regenerative treatment of articular cartilage. Compared to conventional single cells, 3D cell culture is artificially created an environment similar to a living body in vitro so that all cells collectively, a cell culture model that allows growth or interaction with the environment. Therefore, the findings of this study indicate that enhancement of treatment efficiency of stem cells caused by potential of survival and proliferation of hASC spheroid in Osteoarthritis. In conclusion, spheroid positive subpopulation of hASCs has high cell proliferation and survival but not apoptosis and cell death potential, which may contribute to successful cartilage regeneration and the development of stem cell therapies in the future.Objectives:Studied for 3D spheroids to investigate the mechanism of enhancement of survival and proliferationof hASC (human adipose stem cells) spheroid, which may contribute to successful improvement of therapeutic efficacy of stem cells.Methods:Cell isolation and culture / 3D cell culture dish preparation / hASCs culture on 3D cell culture dish / Real-time PCR analysis / Western blotting / Alcian blue staining / ACLT + MM (Anterior cruciate ligament transection with Medial meniscectomy) model / In vivo fluorescence for cell tracking / In vivo effects of spheroids in OA joint / Histological analysis / Enzyme-linked immunosorbent assay (ELISA) results for inflamma -tory cytokines in rat synovial fluid / Statistical AnalysisResults:In order to see how the spheroid showed more residual than single, and how effective it was in actual cartilage regeneration, the result of paraffin tissues were confirmed by safranin O staining for each condition. The tendency of cartilage regeneration efficiency was good for spheroid. Although the differences between the single and spheroid groups were small, they reaffirmed that they could somewhat protect cartilage and help regeneration treatment. However, immunohistochemistry of HN(Human nucleic antigen) staining showed that cells of single and spheroid were not observed in the wound but disappeared by the paracrine effect.Conclusion:Spheroids do not exhibit differentiation characteristics, but they could be seen as a result of expression of related genes such as Bax, Bcl-XL and Alcian blue staining. Spheroids tend to have low potential of cell death rather than proliferation and reduction in the proliferation. So, we conclude the fact that instead of hASCs going directly to the surgical site to regenerate cartilage, they can help catrilage regeneration.Acknowledgments:This research was supported by the National Research Foundation of Korea (NRF-2019R1H1A2039685 and 2019R1I1A1A01043778).Disclosure of Interests:None declared

scholarly journals Comparison of Blood Bacterial Communities in Periodontal Health and Periodontal Disease

2021 ◽  
Vol 10 ◽  
Author(s):  
David C. Emery ◽  
Tanya L. Cerajewska ◽  
Joon Seong ◽  
Maria Davies ◽  
Alex Paterson ◽  
...  
Keyword(s):  
Periodontal Disease ◽  
Periodontal Diseases ◽  
Bacterial Species ◽  
Oral Bacteria ◽  
Bacterial Cells ◽  
Periodontal Health ◽  
True Nature ◽  
Bacterial Dna ◽  
Total Dna ◽  
Microbiome Data

The use of Next Generation Sequencing (NGS) techniques has generated a wide variety of blood microbiome data. Due to the large variation in bacterial DNA profiles between studies and the likely high concentrations of cell-free bacterial DNA in the blood, it is still not clear how such microbiome data relates to viable microbiota. For these reasons much remains to be understood about the true nature of any possible healthy blood microbiota and of bacteraemic events associated with disease. The gut, reproductive tracts, skin, and oral cavity are all likely sources of blood-borne bacteria. Oral bacteria, especially those associated with periodontal diseases, are also commonly associated with cardiovascular diseases such as infective endocarditis, and also have been linked to rheumatoid arthritis and Alzheimer’s disease. Periodontal treatment, dental probing, and toothbrushing have been shown to cause transient bacteraemia and oral bacteria from the phyla Firmicutes (e.g. Streptococci) and Bacteroidetes (e.g. Porphyromonas) are found in cardiovascular lesions (CVD). Many studies of blood bacterial DNA content however, find Proteobacteria DNA to be the dominant microbiome component, suggesting a gut origin. Most studies of this type use total DNA extracted from either whole blood or blood fractions, such as buffy coat. Here, using a method that purifies DNA from intact bacterial cells only, we examined blood donated by those with active, severe periodontitis and periodontally healthy controls and show that 43–52% of bacterial species in blood are classified as oral. Firmicutes, consisting largely of members of the Streptococcus mitis group and Staphylococcus epidermidis, were predominant at 63.5% of all bacterial sequences detected in periodontal health and, little changed at 66.7% in periodontitis. Compared to studies using total DNA Proteobacteria were found here at relatively low levels in blood at 13.3% in periodontitis and 17.6% in health. This study reveals significant phylogenetic differences in blood bacterial population profiles when comparing periodontal health to periodontal disease cohorts.

Process Description of an Unconventional Biofilm Formation by Bacterial Cells Autoagglutinating Through Sticky, Long, and Peritrichate Nanofibers

2019 ◽  
Author(s):  
Yoshihide Furuichi ◽  
Shogo Yoshimoto ◽  
Tomohiro Inaba ◽  
Nobuhiko Nomura ◽  
Katsutoshi Hori
Keyword(s):  
Formation Process ◽  
Biofilm Formation ◽  
Extracellular Polymeric Substances ◽  
Waste Treatment ◽  
Large Cell ◽  
Dlvo Theory ◽  
Bacterial Cells ◽  
Chemical Production ◽  
Discontinuous Surface ◽  
Cell Cell

<p></p><p>Biofilms are used in environmental biotechnologies including waste treatment and environmentally friendly chemical production. Understanding the mechanisms of biofilm formation is essential to control microbial behavior and improve environmental biotechnologies. <i>Acinetobacter </i>sp. Tol 5 autoagglutinate through the interaction of the long, peritrichate nanofiber protein AtaA, a trimeric autotransporter adhesin. Using AtaA, without cell growth or the production of extracellular polymeric substances, Tol 5 cells quickly form an unconventional biofilm. In this study, we investigated the formation process of this unconventional biofilm, which started with cell–cell interactions, proceeded to cell clumping, and led to the formation of large cell aggregates. The cell–cell interaction was described by DLVO theory based on a new concept, which considers two independent interactions between two cell bodies and between two AtaA fiber tips forming a virtual discontinuous surface. If cell bodies cannot collide owing to an energy barrier at low ionic strengths but approach within the interactive distance of AtaA fibers, cells can agglutinate through their contact. Cell clumping proceeds following the cluster–cluster aggregation model, and an unconventional biofilm containing void spaces and a fractal nature develops. Understanding its formation process would extend the utilization of various types of biofilms, enhancing environmental biotechnologies.</p><p></p>

Antibiotics Application Strategies to Control Biofilm Formation in Pathogenic Bacteria

2020 ◽  
Vol 21 (4) ◽  
pp. 270-286 ◽  
Author(s):  
Fazlurrahman Khan ◽  
Dung T.N. Pham ◽  
Sandra F. Oloketuyi ◽  
Young-Mog Kim
Keyword(s):  
Biofilm Formation ◽  
Pathogenic Bacteria ◽  
Extracellular Polymeric Substances ◽  
Quorum Quenching ◽  
Resistance Mechanisms ◽  
Bacterial Biofilm ◽  
Bacterial Cells ◽  
High Concentration ◽  
Biofilm Inhibition ◽  
Abiotic Surfaces

Background: The establishment of a biofilm by most pathogenic bacteria has been known as one of the resistance mechanisms against antibiotics. A biofilm is a structural component where the bacterial community adheres to the biotic or abiotic surfaces by the help of Extracellular Polymeric Substances (EPS) produced by bacterial cells. The biofilm matrix possesses the ability to resist several adverse environmental factors, including the effect of antibiotics. Therefore, the resistance of bacterial biofilm-forming cells could be increased up to 1000 times than the planktonic cells, hence requiring a significantly high concentration of antibiotics for treatment. Methods: Up to the present, several methodologies employing antibiotics as an anti-biofilm, antivirulence or quorum quenching agent have been developed for biofilm inhibition and eradication of a pre-formed mature biofilm. Results: Among the anti-biofilm strategies being tested, the sub-minimal inhibitory concentration of several antibiotics either alone or in combination has been shown to inhibit biofilm formation and down-regulate the production of virulence factors. The combinatorial strategies include (1) combination of multiple antibiotics, (2) combination of antibiotics with non-antibiotic agents and (3) loading of antibiotics onto a carrier. Conclusion: The present review paper describes the role of several antibiotics as biofilm inhibitors and also the alternative strategies adopted for applications in eradicating and inhibiting the formation of biofilm by pathogenic bacteria.

scholarly journals Characteristic Microbiomes Correlate with Polyphosphate Accumulation of Marine Sponges in South China Sea Areas

2019 ◽  
Vol 8 (1) ◽  
pp. 63
Author(s):  
Huilong Ou ◽  
Mingyu Li ◽  
Shufei Wu ◽  
Linli Jia ◽  
Russell T. Hill ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Inorganic Phosphorus ◽  
Marine Sponges ◽  
Strongly Correlated ◽  
Mantel Tests ◽  
Low Concentrations ◽  
Enrichment Rate ◽  
Total Dna ◽  
Polyphosphate Accumulation ◽  
Sequence Similarities

Some sponges have been shown to accumulate abundant phosphorus in the form of polyphosphate (polyP) granules even in waters where phosphorus is present at low concentrations. But the polyP accumulation occurring in sponges and their symbiotic bacteria have been little studied. The amounts of polyP exhibited significant differences in twelve sponges from marine environments with high or low dissolved inorganic phosphorus (DIP) concentrations which were quantified by spectral analysis, even though in the same sponge genus, e.g., Mycale sp. or Callyspongia sp. PolyP enrichment rates of sponges in oligotrophic environments were far higher than those in eutrophic environments. Massive polyP granules were observed under confocal microscopy in samples from very low DIP environments. The composition of sponge symbiotic microbes was analyzed by high-throughput sequencing and the corresponding polyphosphate kinase (ppk) genes were detected. Sequence analysis revealed that in the low DIP environment, those sponges with higher polyP content and enrichment rates had relatively higher abundances of cyanobacteria. Mantel tests and canonical correspondence analysis (CCA) examined that the polyP enrichment rate was most strongly correlated with the structure of microbial communities, including genera Synechococcus, Rhodopirellula, Blastopirellula, and Rubripirellula. About 50% of ppk genes obtained from the total DNA of sponge holobionts, had above 80% amino acid sequence similarities to those sequences from Synechococcus. In general, it suggested that sponges employed differentiated strategies towards the use of phosphorus in different nutrient environments and the symbiotic Synechococcus could play a key role in accumulating polyP.

scholarly journals Stochasticity in Colonial Growth Dynamics of Individual Bacterial Cells

2013 ◽  
Vol 79 (7) ◽  
pp. 2294-2301 ◽  
Author(s):  
Konstantinos P. Koutsoumanis ◽  
Alexandra Lianou
Keyword(s):  
Kinetic Parameters ◽  
Single Cells ◽  
Growth Curves ◽  
Growth Dynamics ◽  
Time Lapse ◽  
Initial Population ◽  
Bacterial Cells ◽  
Stochastic Growth ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTConventional bacterial growth studies rely on large bacterial populations without considering the individual cells. Individual cells, however, can exhibit marked behavioral heterogeneity. Here, we present experimental observations on the colonial growth of 220 individual cells ofSalmonella entericaserotype Typhimurium using time-lapse microscopy videos. We found a highly heterogeneous behavior. Some cells did not grow, showing filamentation or lysis before division. Cells that were able to grow and form microcolonies showed highly diverse growth dynamics. The quality of the videos allowed for counting the cells over time and estimating the kinetic parameters lag time (λ) and maximum specific growth rate (μmax) for each microcolony originating from a single cell. To interpret the observations, the variability of the kinetic parameters was characterized using appropriate probability distributions and introduced to a stochastic model that allows for taking into account heterogeneity using Monte Carlo simulation. The model provides stochastic growth curves demonstrating that growth of single cells or small microbial populations is a pool of events each one of which has its own probability to occur. Simulations of the model illustrated how the apparent variability in population growth gradually decreases with increasing initial population size (N0). For bacterial populations withN0of >100 cells, the variability is almost eliminated and the system seems to behave deterministically, even though the underlying law is stochastic. We also used the model to demonstrate the effect of the presence and extent of a nongrowing population fraction on the stochastic growth of bacterial populations.

scholarly journals LARYNGOTRACHEITIS VIRUS IN CHICKENS

1967 ◽  
Vol 125 (3) ◽  
pp. 409-428 ◽  
Author(s):  
Betsy G. Bang ◽  
Frederik B. Bang
Keyword(s):  
Alcian Blue ◽  
Plasma Cells ◽  
Giant Cells ◽  
Functional Restoration ◽  
Nasal Fossa ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Cell Nuclei ◽  
Histochemical Change ◽  
Laryngotracheitis Virus

Infectious laryngotracheitis can be produced in chickens as an experimental model of severe nonfatal rhinitis and sinusitis. Inoculated intranasally into unanesthetized baby chicks it remains limited to the nasal fossa, produces acute desquamation of all nasal epithelia, results in functional recovery of the respiratory epithelium, but leaves important residual abnormalities. From the earliest recognizable lesions through 4½ months' convalescence, the principal changes are as follows: 1. Initial lesions, or small syncytia of intranuclear "inclusions", first identifiable in the mucociliated cells of the shallowest portion of the epithelium at about 21 hr postinoculum (the inner surface of the maxillary conchal scroll). 2. Acute sloughing, (about 3 to 7 days), marked by: (a) spread of lesions from cell to cell via multinucleated "giant cells" which progressively slough and desquamate respiratory, olfactory, and sinus epithelia, epithelial neural elements and blood vessels; (b) appearance of numbers of eosinophilic leukocytes along the basement membrane at the sites of lesions just previous to sloughing; intensive infiltration of the submucosa with small lymphocytes after sloughing begins; (c) histochemical change in the intracellular mucus of the cells which comprise the syncytia: this mucus stains with Alcian blue alone when stained with AB-PAS; and (d) all cartilages of the maxillary conchae become flaccid, and the cell nuclei and matrix lose both basophilic and Alcian blue staining properties, effects which recede by about the 8th day. 3. Repair (about 8 to 21 days), marked by rapid initial spread of a sheet of epithelial cells over the infiltrated subrmucosa, appearance of numbers of plasma cells circulating in the tissues, formation of encapsulated secondary nodules, and mucosal adhesions. 4. Convalescence (about 1 to 4½ months when experiments terminated), marked by functional restoration of the mucociliary lining of the nasal fossa. However, at 4½ months eight specimens all show complete metaplasia of the olfactory organ (end nerves, supporting cells, and glands of Bowman) to mucociliated epithelium, all show abnormal formation and alignment of mucous acini, and about 50% have severe persistent sinusitis.

Histochemical observations on the tegumentary epithelium and interproglottidal glands of Moniezia expansa (Rud., 1805) (Cestoda, Cyclophyllidea)

Parasitology ◽  
1969 ◽  
Vol 59 (3) ◽  
pp. 505-518 ◽  
Author(s):  
R. E. Howells ◽  
D. A. Erasmus
Keyword(s):  
Alcian Blue ◽  
Regional Differences ◽  
Succinic Dehydrogenase ◽  
Neck Region ◽  
Secretory Activity ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Light Microscope Level ◽  
Gland Cells ◽  
Moniezia Expansa

Regional differences in the tegumentary tissue of Moniezia expansa, as revealed at the light-microscope level by histological and histochemical techniques, are described and evidence for secretory activity by the interproglottidal glands is presented.In very immature proglottides the interproglottidal glands are at the ‘precryptic’ stage. Gland cells may be differentiated from other tegumentary cells by their high RNA content and in certain gland cells the presence of an alcian blue staining material.In mature proglottides the glands consist of rosette-like clusters of cells around crypt-like intuckings of the tegument. Two types of cells are found in the gland, small alcian blue-staining cells which are most numerous in the neck region of the crypt, and larger cells, the predominant gland cells, which do not stain with alcian blue but possess non-specific esterase activity. No other tegumentary cells in Moniezia exhibit this activity. Esterase and phosphatase activity is found in the tegument and crypt of the glands and in the interproglottidal folds.The non-enzyme histochemistry confirms and extends the observations of previous workers.Cytochrome oxidase and succinic dehydrogenase were detected in the tegumentary cells and tegument. Very strong reactions were given in the neck and scolex, with a progressive diminution of activity posteriorly along the strobila. Very low activities were recorded in the tegument of the glands.

Dual action of sonic hedgehog on chondrocyte hypertrophy:retrovirus mediated ectopic sonic hedgehog expression in limb bud micromass culture induces novel cartilage nodules that are positive for alkaline phosphatase and type X collagen

1997 ◽  
Vol 110 (21) ◽  
pp. 2691-2701 ◽  
Author(s):  
N.S. Stott ◽  
C.M. Chuong
Keyword(s):  
Alkaline Phosphatase ◽  
Gene Family ◽  
Alcian Blue ◽  
Sonic Hedgehog ◽  
Ectopic Expression ◽  
Limb Bud ◽  
Blue Staining ◽  
Alcian Blue Staining ◽  
Type X Collagen ◽  
Chondrocyte Maturation

Members of the vertebrate hedgehog gene family (HH) are involved in patterning and modulation of differentiation. Recently it has been shown that ectopic expression of HH gene family members in vivo blocks chondrocyte maturation through activation of a parathyroid hormone related peptide (PTHrP) dependent negative regulatory loop in the perichondrium. However, the direct effect of HH on chondrocyte maturation has not been tested. Here, we studied the effect of retroviral overexpression of the chicken sonic hedgehog gene (Shh) on the growth and maturation of limb bud cells in micromass cultures. Shh is neither expressed nor required for the initiation of cellular condensation in normal micromass cultures. With Shh over-expression, micromass cultures developed novel tightly whorled nodules in addition to the normal Alcian Blue positive cartilage nodules. We characterized the new nodules and showed that they are strongly positive for alkaline phosphatase, enriched in type X collagen and weakly positive for Alcian Blue staining. Shh overexpression also increased cell proliferation, but this cannot account for the formation of the new nodules. This current study shows that misexpression of Shh in in vitro chondrogenic cultures promotes characteristics of hypertrophic chondrocytes. Thus HH has two complementary functions; a direct positive effect on chondrocyte hypertrophy in the absence of PTHrP pathway, and an indirect negative feedback loop through PTHrP to prevent other less differentiated chondrocytes from becoming hypertrophic. These two complementary actions of HH coordinate the progression of cartilage maturation.

Branching morphogenesis in the avian lung: electron microscopic studies using cationic dyes

Development ◽  
1986 ◽  
Vol 94 (1) ◽  
pp. 189-205
Author(s):  
Betty C. Gallagher
Keyword(s):  
Tannic Acid ◽  
Basal Lamina ◽  
Branching Morphogenesis ◽  
Ruthenium Red ◽  
Interparticle Spacing ◽  
Blue Staining ◽  
Mouse Lung ◽  
Alcian Blue Staining ◽  
Electron Microscopic ◽  
Avian Lung

The developing chick lung was examined in the electron microscope for intimate cell contacts between epithelium and mesenchyme, discontinuities in the basal lamina and substructure of the basement membrane. Cell filopodia were seen which crossed the basal lamina from both the epithelial and the mesenchymal cells. Ruthenium red and tannic acid staining of the basal lamina of the chick lung showed it to be thin and sometimes discontinuous at the tips compared to the more substantial basal lamina in the interbud areas. The bilaminar distribution of particles seen with ruthenium red is similar to those seen in the cornea and lens. With tannic acid staining, filaments could be seen which crossed the lamina lucida and connected with the lamina densa. Spikes perpendicular to the basal lamina were sometimes seen with a periodicity of approximately 110 nm. Alcian blue staining revealed structure similar to that seen by ruthenium red staining in the salivary and mammary glands, although the interparticle spacing was closer. Collagen was located in areas of morphogenetic stability, as has been seen by other investigators in different tissues. Collagen was coated with granules (probably proteoglycan) at periodic intervals when stained with ruthenium red. The fibrils were oriented circumferentially around the mesobronchus and were assumed to continue into the bud, but the fibres curve laterally at the middle of a bud. This orientation is opposite to that seen by another investigator in the mouse lung. In general, the observations made in the avian lung are similar to those seen in branching mammalian tissue. It is likely, therefore, that the chick lung uses strategies in its morphogenesis that are similar to those that have been elucidated previously in developing mammalian organs.

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