The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm

0491213 - ÚPT 2019 RIV GB eng J - Journal Article
Hrubanová, Kamila - Nebesářová, Jana - Růžička, F. - Krzyžánek, Vladislav
The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm.
Micron. Roč. 110, JUL (2018), s. 28-35. ISSN 0968-4328. E-ISSN 1878-4291
R&D Projects: GA ČR GA17-15451S; GA MŠMT(CZ) LO1212; GA MŠMT(CZ) LM2015062; GA MŠMT ED0017/01/01
Institutional support: RVO:68081731 ; RVO:60077344
Keywords : cryo-SEM * high-pressure freezing (HPF) * freeze-fracturing * biofilm * candida * staphylococcus
OECD category: Microbiology; Reproductive biology (medical aspects to be 3) (BC-A)
Impact factor: 1.530, year: 2018

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.
Permanent Link: http://hdl.handle.net/11104/0285253