In this study we present an innovative method for the preparation of fully hydrated samples of microbial biofilms of cultures Staphylococcus epidermidis, Candida parapsilosis and Candida albicans. Cryo-scanning electron microscopy (cryo-SEM) and high-pressure freezing (HPF) rank among cutting edge techniques in the electron microscopy of hydrated samples such as biofilms. However, the combination of these techniques is not always easily applicable. Therefore, we present a method of combining high-pressure freezing using EM PACT2 (Leica Microsystems), which fixes hydrated samples on small sapphire discs, with a high resolution SEM equipped with the widely used cryo-preparation system ALTO 2500 (Gatan). Using a holder developed in house, a freeze-fracturing technique was applied to image and investigate microbial cultures cultivated on the sapphire discs. In our experiments, we focused on the ultrastructure of the extracellular matrix produced during cultivation and the relationships among microbial cells in the biofilm. The main goal of our investigations was the detailed visualization of areas of the biofilm where the microbial cells adhere to the substrate/surface. We show the feasibility of this technique, which is clearly demonstrated in experiments with various freeze-etching times.

• MeSH
• biofilmy MeSH
• Candida albicans ultrastruktura   MeSH
• Candida parapsilosis ultrastruktura   MeSH
• elektronová kryomikroskopie metody   MeSH
• extracelulární matrix ultrastruktura   MeSH
• mikroskopie elektronová rastrovací metody   MeSH
• mrazové lámání metody   MeSH
• Staphylococcus epidermidis ultrastruktura   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The effects of acoustic waves on membrane structures, and any resulting consequences of this treatment on membrane subunit structures, remain poorly understood, as are the principals of related clinical effects. With a focus on morphological changes in the nuclear envelope, the current study presents detailed observations of membrane structures exposed to therapeutic ultrasound. Ultrasound treatment most commonly resulted in distinct changes in the distribution of nuclear pore complexes (NPCs) and mean NPC number per unit area after 30 min of repair, as well as alterations in NPC diameters on the protoplasmic face of fractured nuclear membranes after 10 min of repair. The greatest effects of ultrasound on nuclear envelope structure and NPCs were not to appear immediately, but became evident after repair processes were initiated. Results from the current study may contribute to the general view on the biophysical effects of therapeutic ultrasound on cell morphology and, particularly, the understanding of this effect in relation to the nuclear envelope.

• MeSH
• elektronová mikroskopie metody   využití   MeSH
• HL-60 buňky cytologie   ultrasonografie   MeSH
• intracelulární membrány ultrasonografie   MeSH
• jaderný obal * ultrasonografie   ultrastruktura   MeSH
• jaderný pór ultrastruktura   MeSH
• komplex proteinů jaderného póru * ultrastruktura   MeSH
• kultivační techniky metody   využití   MeSH
• leptání mrazem metody   využití   MeSH
• lidé MeSH
• mikrofotografie MeSH
• struktury buněčného jádra ultrasonografie   MeSH
• ultrasonografie metody   škodlivé účinky   využití   MeSH
• ultrazvuková terapie * metody   škodlivé účinky   využití   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Microtubular and actin cytoskeletons were investigated in the lipophilic yeast Malassezia pachydermatis by fluorescence and electron microscopy. To detect microtubules by indirect immunofluorescence using monoclonal anti-tubulin antibody, a prolonged incubation with lysing enzymes was necessary due to its very thick cell wall. Cytoplasmic microtubules were detected in interphase and a spindle with astral microtubules was seen in M-phase. The disintegration of cytoplasmic microtubules and migration of the nucleus to the bud before mitosis were characteristic features of the basidiomycetous yeast Malassezia pachydermatis. The visualisation of F-actin structures (patches, cables and cytokinetic rings) by fluorescence microscopy using both monoclonal anti-actin antibody and rhodamine-phalloidin failed, but actin was detected by electron microscopy with immunogold labelling. Clusters of gold particles indicating actin structures were detected at the plasma membrane of cells with unique cortical ultrastructural features characteristic of the genus Malassezia. A possible association of these with the actin cytoskeleton is suggested.

• MeSH
• aktiny ultrastruktura   MeSH
• biologické modely MeSH
• buněčný cyklus MeSH
• cytoskelet metabolismus   ultrastruktura   MeSH
• financování organizované MeSH
• fluorescenční mikroskopie MeSH
• imunohistochemie MeSH
• Malassezia ultrastruktura   MeSH
• mikrotomie MeSH
• mikrotubuly ultrastruktura   MeSH
• mrazové lámání MeSH

• MeSH
• buněčná membrána patologie   MeSH
• finanční podpora výzkumu jako téma MeSH
• leptání mrazem metody   využití   MeSH
• nádorové buňky kultivované cytologie   MeSH
• pilotní projekty MeSH
• ultrazvuk škodlivé účinky   MeSH
• Publikační typ
• kongresy MeSH

Under the light microscope the chitin-protein layer of egg-shells in ascarids appears to be a regular, hyaline and nonstructural layer of 1.5 to 2.00 microns in thickness. The outer uterine layer is usually removed during the preparation. The lipid (ascaroside) layer covers the inner surface of the chitinous layer and seems to be irregularly undulated and regularly thick over the whole surface, with the thickness up to 1 micron. In electron micrographs the fibrous structure of the lipid layer is not evident as a rule. This is probably due to washing the lipids away from this layer during the dehydration of deeper layers of egg-shells that are imperfectly fixed with glutaraldehyde. A very low permeability of the egg-shells is typical of geohelminth eggs. The layer lipid shows a distinct lamellate structure only after a prolonged fixation with osmium at higher temperature. This is supported by the studies using the method of freeze-fracturing.

• MeSH
• Ascaris ultrastruktura   MeSH
• chitin MeSH
• elektronová mikroskopie MeSH
• lipidy MeSH
• mrazové lámání MeSH
• ovum ultrastruktura   MeSH
• zvířata MeSH
• Check Tag
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• mezibuněčné spoje MeSH
• morčata MeSH
• mrazové lámání MeSH
• vnitřní ucho MeSH
• Check Tag
• morčata MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• otorinolaryngologie