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

J 2018

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

HRUBANOVA, Kamila, Jana NEBESAROVA, Filip RŮŽIČKA and Vladislav KRZYZANEK

Basic information

Original name

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

Authors

HRUBANOVA, Kamila (203 Czech Republic), Jana NEBESAROVA (203 Czech Republic), Filip RŮŽIČKA (203 Czech Republic, belonging to the institution) and Vladislav KRZYZANEK (203 Czech Republic, guarantor)

Edition

Micron, OXFORD, PERGAMON-ELSEVIER SCIENCE LTD, 2018, 0968-4328

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10606 Microbiology

Country of publisher

United Kingdom of Great Britain and Northern Ireland

Confidentiality degree

není předmětem státního či obchodního tajemství

Impact factor

Impact factor: 1.530

RIV identification code

RIV/00216224:14110/18:00104166

Organization unit

Faculty of Medicine

DOI

http://dx.doi.org/10.1016/j.micron.2018.04.006

UT WoS

000435048000004

Keywords in English

cryo-SEM; High-pressure freezing (HPF); Freeze-fracturing; Biofilm; Candida; Staphylococcus

Abstract

V originále

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.

Links

NV16-29916A, research and development project

Name: Využití bakteriofágů v léčbě nozokomiálních infekcí spojených s multirezistencí či tvorbou biofilmu

Citovat

HRUBANOVA, Kamila, Jana NEBESAROVA, Filip RŮŽIČKA and Vladislav KRZYZANEK. The innovation of cryo-SEM freeze-fracturing methodology demonstrated on high pressure frozen biofilm. 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.

@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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