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@article{1454496,
author = {Hrubanova, Kamila and Nebesarova, Jana and Růžička, Filip and Krzyzanek, Vladislav},
article_location = {OXFORD},
article_number = {JUL 2018},
doi = {http://dx.doi.org/10.1016/j.micron.2018.04.006},
keywords = {cryo-SEM; High-pressure freezing (HPF); Freeze-fracturing; Biofilm; Candida; Staphylococcus},
language = {eng},
issn = {0968-4328},
journal = {Micron},
title = {The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm},
volume = {110},
year = {2018}
}
TY - JOUR
ID - 1454496
AU - Hrubanova, Kamila - Nebesarova, Jana - Růžička, Filip - Krzyzanek, Vladislav
PY - 2018
TI - The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm
JF - Micron
VL - 110
IS - JUL 2018
SP - 28-35
EP - 28-35
PB - PERGAMON-ELSEVIER SCIENCE LTD
SN - 09684328
KW - cryo-SEM
KW - High-pressure freezing (HPF)
KW - Freeze-fracturing
KW - Biofilm
KW - Candida
KW - Staphylococcus
N2 - 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 micro-scopy (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.
ER -
HRUBANOVA, Kamila, Jana NEBESAROVA, Filip RŮŽIČKA and Vladislav KRZYZANEK. The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm. \textit{Micron}. OXFORD: PERGAMON-ELSEVIER SCIENCE LTD, 2018, vol.~110, JUL 2018, p.~28-35. ISSN~0968-4328. Available from: https://dx.doi.org/10.1016/j.micron.2018.04.006.
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