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The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm
K. Hrubanova, J. Nebesarova, F. Ruzicka, V. Krzyzanek,
Language English Country England, Great Britain
Document type Journal Article, Research Support, Non-U.S. Gov't
Grant support
NV16-29916A
MZ0
CEP Register
- MeSH
- Biofilms MeSH
- Candida albicans ultrastructure MeSH
- Candida parapsilosis ultrastructure MeSH
- Cryoelectron Microscopy methods MeSH
- Extracellular Matrix ultrastructure MeSH
- Microscopy, Electron, Scanning methods MeSH
- Freeze Fracturing methods MeSH
- Staphylococcus epidermidis ultrastructure MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
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.
Faculty of Science University of South Bohemia Ceske Budejovice Czech Republic
Institute of Scientific Instruments of the Czech Academy of Sciences Brno Czech Republic
References provided by Crossref.org
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- $a 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.
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