scholarly journals The MNN2 Gene Knockout Modulates the Antifungal Resistance of Biofilms of Candida glabrata

Biomolecules ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 130 ◽  
Author(s):  
Celia F. Rodrigues ◽  
Diana Vilas Boas ◽  
Ken Haynes ◽  
Mariana Henriques
Keyword(s):  
Cell Walls ◽  
Candida Glabrata ◽  
Laser Scanning ◽  
Reference Strain ◽  
Gene Knockout ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Drug Treatments ◽  
Biomass Reduction ◽  
Biofilm Matrix

Candida glabrata biofilms are recognized to have high resistance to antifungals. In order to understand the effect of mannans in the resistance profile of C. glabrata mature biofilms, C. glabrata Δmnn2 was evaluated. Biofilm cell walls were analysed by confocal laser scanning microscopy (CLSM) and their susceptibility was assessed for fluconazole, amphotericin B, caspofungin, and micafungin. Crystal violet and Alcian Blue methods were performed to quantify the biomass and the mannans concentration in the biofilm cells and matrices, respectively. The concentration of β-1,3 glucans was also measured. No visible differences were detected among cell walls of the strains, but the mutant had a high biomass reduction, after a drug stress. When compared with the reference strain, it was detected a decrease in the susceptibility of the biofilm cells and an increase of β-1,3 glucans in the C. glabrata Δmnn2. The deletion of the MNN2 gene in C. glabrata induces biofilm matrix and cell wall variabilities that increase the resistance to the antifungal drug treatments. The rise of β-1,3 glucans appears to have a role in this effect.

scholarly journals High Adhesion and Increased Cell Death Contribute to Strong Biofilm Formation in Klebsiella pneumoniae

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 277
Author(s):  
Siddhi Desai ◽  
Kinjal Sanghrajka ◽  
Devarshi Gajjar
Keyword(s):  
Cell Death ◽  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Confocal Laser ◽  
Bacterial Cells ◽  
High Adhesion ◽  
Biofilm Matrix

Klebsiella pneumoniae (Kp), is a frequent cause of hospital and community-acquired infections and WHO had declared it as a “priority pathogen”. Biofilm is a major virulence factor of Kp and yet the mechanism of strong biofilm formation in Kp is unclear. A key objective of the present study is to investigate the differences between strong and weak biofilms formed by clinical isolates of Kp on various catheters and in different media conditions and to identify constituents contributing to strong biofilm formation. Quantification of matrix components (extracellular DNA (eDNA), protein, exopolysaccharides (EPS), and bacterial cells), confocal laser scanning microscopy (CLSM), field emission gun scanning electron microscopy (FEG-SEM) and flow-cytometry analysis were performed to compare strong and weak biofilm matrix. Our results suggest increased biofilm formation on latex catheters compared to silicone and silicone-coated latex catheters. Higher amounts of eDNA, protein, EPS, and dead cells were observed in the strong biofilm of Kp. High adhesion capacity and cell death seem to play a major role in formation of strong Kp biofilms. The enhanced eDNA, EPS, and protein in the biofilm matrix appear as a consequence of increased cell death.

scholarly journals Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems

AoB Plants ◽  
2020 ◽  
Vol 12 (4) ◽  
Author(s):  
Peter Kitin ◽  
Satoshi Nakaba ◽  
Christopher G Hunt ◽  
Sierin Lim ◽  
Ryo Funada
Keyword(s):  
Cell Walls ◽  
Laser Scanning ◽  
Cell Types ◽  
Plant Evolution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Chemical Information ◽  
Wide Field ◽  
Tissue Preparation ◽  
Plant Structure

Abstract Investigating plant structure is fundamental in botanical science and provides crucial knowledge for the theories of plant evolution, ecophysiology and for the biotechnological practices. Modern plant anatomy often targets the formation, localization and characterization of cellulosic, lignified or suberized cell walls. While classical methods developed in the 1960s are still popular, recent innovations in tissue preparation, fluorescence staining and microscopy equipment offer advantages to the traditional practices for investigation of the complex lignocellulosic walls. Our goal is to enhance the productivity and quality of microscopy work by focusing on quick and cost-effective preparation of thick sections or plant specimen surfaces and efficient use of direct fluorescent stains. We discuss popular histochemical microscopy techniques for visualization of cell walls, such as autofluorescence or staining with calcofluor, Congo red (CR), fluorol yellow (FY) and safranin, and provide detailed descriptions of our own approaches and protocols. Autofluorescence of lignin in combination with CR and FY staining can clearly differentiate between lignified, suberized and unlignified cell walls in root and stem tissues. Glycerol can serve as an effective clearing medium as well as the carrier of FY for staining of suberin and lipids allowing for observation of thick histological preparations. Three-dimensional (3D) imaging of all cell types together with chemical information by wide-field fluorescence or confocal laser scanning microscopy (CLSM) was achieved.

Cytological observation of the infection process of Venturia carpophila on peach leaves

Plant Disease ◽  
2021 ◽  
Author(s):  
Yang Zhou ◽  
Lei Zhang ◽  
Chuan-Ya Ji ◽  
Chingchai Chaisiri ◽  
Liangfen Yin ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Laser Scanning ◽  
Infection Process ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Post Inoculation ◽  
Transmission Electron ◽  
Leaf Surfaces ◽  
Germ Tubes

Peach scab caused by Venturia carpophila, is one of the most destructive fungal diseases of peach worldwide, which seriously affects the peach production. Up to date, the infection process and pathogenesis of V. carpophila on peach remain unclear. Here, we present the infection behaviour of V. carpophila at the ultrastructural and cytological levels in peach leaves with combined microscopic investigations (e.g., light microscopy, confocal laser scanning microscopy, scanning electron microscopy and transmission electron microscopy). V. carpophila germinated at the tip of conidia and produced short germ tubes on peach leaf surfaces at 2 days post-inoculation (dpi). At 3 dpi, swollen tips of germ tubes differentiated into appressoria. At 5 dpi, penetration pegs produced by appressoria broke through the cuticle layer, and then differentiated into thick sub-cuticular hyphae in the pectin layer of the epidermal cell walls. At 10 dpi, the sub-cuticular hyphae extensively colonized in the pectin layer. The primary hyphae ramified into secondary hyphae and proliferated along with the incubation. At 15 dpi, the sub-cuticular hyphae divided laterally to form stromata between the cuticle layer and the cellulose layer of the epidermal cells. At 30 dpi, conidiophores developed from the sub-cuticular stromata. Finally, abundant conidiophores and new conidia appeared on leaf surfaces at 40 dpi. These results provide useful information for further understanding the V. carpophila pathogenesis.

scholarly journals Effect of Phenol Formaldehyde Resin Penetration on the Quasi-Static and Dynamic Mechanics of Wood Cell Walls Using Nanoindentation

Nanomaterials ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 1409 ◽  
Author(s):  
Xinzhou Wang ◽  
Xuanzong Chen ◽  
Xuqin Xie ◽  
Zhurun Yuan ◽  
Shaoxiang Cai ◽  
...  
Keyword(s):  
Cell Walls ◽  
Laser Scanning ◽  
Phenol Formaldehyde ◽  
Phenol Formaldehyde Resin ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Formaldehyde Resin ◽  
Masson Pine ◽  
Crystallinity Degree ◽  
Modified Wood

To evaluate the effects of phenol formaldehyde (PF) resin modification on wood cell walls, Masson pine (Pinus massoniana Lamb.) wood was impregnated with PF resin at the concentrations of 15%, 20%, 25%, and 30%, respectively. The penetration degree of PF resin into wood tracheids was quantitatively determined using confocal laser scanning microscopy (CLSM). The micromechanical properties of the control and PF-modified wood cell walls were then analyzed by the method of quasi-static nanoindentation and dynamic modulus mapping techniques. Results indicated that PF resin with low molecular weight can penetrate deeply into the wood tissues and even into the cell walls. However, the penetration degree decreased accompanying with the increase of penetration depth in wood. Both the quasi-static and dynamic mechanics of wood cell walls increased significantly after modification by the PF resin at the concentration less than 20%. The cell-wall mechanics maintained stable and even decreased as the resin concentration was increased above 20%, resulting from the increasing bulking effects such as the decreased crystallinity degree of cellulose. Furthermore, the mechanics of cell walls in the inner layer was lower than that in the outer layer of PF-modified wood.

scholarly journals The Protective Effect of Staphylococcus epidermidis Biofilm Matrix against Phage Predation

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1076
Author(s):  
Luís D. R. Melo ◽  
Graça Pinto ◽  
Fernando Oliveira ◽  
Diana Vilas-Boas ◽  
Carina Almeida ◽  
...  
Keyword(s):  
Protective Effect ◽  
Staphylococcus Epidermidis ◽  
Laser Scanning ◽  
3D Structure ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Planktonic Cells ◽  
The Matrix ◽  
The Impact ◽  
Biofilm Matrix

Staphylococcus epidermidis is a major causative agent of nosocomial infections, mainly associated with the use of indwelling devices, on which this bacterium forms structures known as biofilms. Due to biofilms’ high tolerance to antibiotics, virulent bacteriophages were previously tested as novel therapeutic agents. However, several staphylococcal bacteriophages were shown to be inefficient against biofilms. In this study, the previously characterized S. epidermidis-specific Sepunavirus phiIBB-SEP1 (SEP1), which has a broad spectrum and high activity against planktonic cells, was evaluated concerning its efficacy against S. epidermidis biofilms. The in vitro biofilm killing assays demonstrated a reduced activity of the phage. To understand the underlying factors impairing SEP1 inefficacy against biofilms, this phage was tested against distinct planktonic and biofilm-derived bacterial populations. Interestingly, SEP1 was able to lyse planktonic cells in different physiological states, suggesting that the inefficacy for biofilm control resulted from the biofilm 3D structure and the protective effect of the matrix. To assess the impact of the biofilm architecture on phage predation, SEP1 was tested in disrupted biofilms resulting in a 2 orders-of-magnitude reduction in the number of viable cells after 6 h of infection. The interaction between SEP1 and the biofilm matrix was further assessed by the addition of matrix to phage particles. Results showed that the matrix did not inactivate phages nor affected phage adsorption. Moreover, confocal laser scanning microscopy data demonstrated that phage infected cells were less predominant in the biofilm regions where the matrix was more abundant. Our results provide compelling evidence indicating that the biofilm matrix can work as a barrier, allowing the bacteria to be hindered from phage infection.

scholarly journals Detecting Bacteria in Wood using a Fluorescent Lipid Probe

1998 ◽  
Vol 6 (7) ◽  
pp. 16-17
Author(s):  
Ying Xiao ◽  
Adya P. Singh ◽  
Robin N. Wakeling
Keyword(s):  
Cell Walls ◽  
Laser Scanning ◽  
Fluorescent Microscopy ◽  
Molecular Probes ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Membrane Probe ◽  
Fungal Hyphae ◽  
Staining Techniques ◽  
Auto Fluorescence

Wood cells have strong autofluorescence in a wide wavelength band due to lignin in the cell walls. The detection of microorganisms in wood is very difficult when using fluorescent microscopy because of interferences. We have recently developed fluorescent staining techniques to differentiate fungal hyphae from wood cell walls (Singh, et al., 1997; Xiao, et al., 1997). This study was aimed at developing fluorescent techniques to visualize bacteria in wood using confocal laser scanning microscopy (CLSM). Nitrobenzoxadiazole glycerophosphoethanolamine (NBD-PE, Molecular Probes), a widely used membrane probe which accords strong fluorescence upon lipids, was compared with glutaraldehyde which had proved useful in our initial attempts to visualize fungal hyphae in wood because of the cell auto fluorescence it causes (Singh et al., 1997).

Micromorphological Characteristics of Compression Wood Degradation in Waterlogged Archaeological Pine Wood

Holzforschung ◽  
1999 ◽  
Vol 53 (4) ◽  
pp. 381-385 ◽  
Author(s):  
Yoon Soo Kim ◽  
Adya P. Singh
Keyword(s):  
Electron Microscopy ◽  
Cell Walls ◽  
Laser Scanning ◽  
Compression Wood ◽  
Outer Part ◽  
Middle Lamella ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Wood Degradation ◽  
Transmission Electron

Summary The degradation characteristics of waterlogged archaeological compression wood excavated in South Korea were examined by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Degradation of Pinus compression wood occurred mainly in the inner part of the S2 layer. In contrast, the outer part of the S2 layer remained relatively intact. CLSM and TEM showed the erosion type of bacterial attack to be dominant in the secondary cell walls of both severe and mild compression wood. However, in some cases the middle lamella was also degraded, which suggests that other forms of microbial attacks, such as bacterial tunnelling, were also present. Bacterial erosion in the severe compression wood was mainly confined to the inner part of the S2 layer whereas in the mild compression wood it also extended into the outer part of the S2 and the S1 layer. The extent of erosion correlated to the differences in the amount and distribution of lignin, particularly in the outer S2 layer between the severe and mild compression wood cells. These features are compared with the degradation of normal Pinus wood.

Microstructure and Quantitative Micromechanical Analysis of Wood Cell–Emulsion Polymer Isocyanate and Urea–Formaldehyde Interphases

2017 ◽  
Vol 23 (3) ◽  
pp. 687-695 ◽  
Author(s):  
Lizhe Qin ◽  
Lanying Lin ◽  
Feng Fu ◽  
Mizi Fan
Keyword(s):  
Cell Wall ◽  
Cell Walls ◽  
Laser Scanning ◽  
Urea Formaldehyde ◽  
Wood Adhesive ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Micromechanical Analysis ◽  
Emulsion Polymer ◽  
Uf Resin

AbstractEmulsion polymer isocyanate (EPI) and urea-formaldehyde (UF) were selected as typical resin systems to investigate the microstructure of wood–adhesive interphases by fluorescence microscopy (FM) and confocal laser scanning microscopy (CLSM). Further, a quantitative micromechanical analysis of the interphases was conducted using nanoindentation. The FM results showed that the UF resin could penetrate the wood to a greater extent than the EPI resin, and that the average penetration depth for these two resin systems was higher in the case of latewood. CLSM allowed visualization of the resin distribution with contrasting colors, showing that the EPI resin could not penetrate the cell wall, whereas UF resin could enter the cell walls. The micromechanical properties of the cell walls were almost unaffected by EPI penetration but were significantly affected by UF penetration, especially in the first cell wall from the glueline. This further confirmed that only cell walls with resin penetration can improve the mechanical properties of the interphase regions.

3-D imaging of cells using a confocal laser scanning microscope and digital image processing

1988 ◽  
Vol 46 ◽  
pp. 96-97
Author(s):  
Thomas M. Jovin ◽  
Michel Robert-Nicoud ◽  
Donna J. Arndt-Jovin ◽  
Thorsten Schormann
Keyword(s):  
Laser Scanning ◽  
Confocal Laser Scanning Microscope ◽  
Laser Scanning Microscopy ◽  
Confocal Laser Scanning ◽  
Confocal Laser ◽  
Scanning Microscope ◽  
Laser Scanning Microscope ◽  
Biochemical Processes ◽  
Adjacent Structures

Light microscopic techniques for visualizing biomolecules and biochemical processes in situ have become indispensable in studies concerning the structural organization of supramolecular assemblies in cells and of processes during the cell cycle, transformation, differentiation, and development. Confocal laser scanning microscopy offers a number of advantages for the in situ localization and quantitation of fluorescence labeled targets and probes: (i) rejection of interfering signals emanating from out-of-focus and adjacent structures, allowing the “optical sectioning” of the specimen and 3-D reconstruction without time consuming deconvolution; (ii) increased spatial resolution; (iii) electronic control of contrast and magnification; (iv) simultanous imaging of the specimen by optical phenomena based on incident, scattered, emitted, and transmitted light; and (v) simultanous use of different fluorescent probes and types of detectors.We currently use a confocal laser scanning microscope CLSM (Zeiss, Oberkochen) equipped with 3-laser excitation (u.v - visible) and confocal optics in the fluorescence mode, as well as a computer-controlled X-Y-Z scanning stage with 0.1 μ resolution.

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