The biofilm-forming microbial species Candida parapsilosis and Staphylococcus epidermidis have been recently linked to serious infections associated with implanted medical devices. We studied microbial biofilms by high resolution scanning electron microscopy (SEM), which allowed us to visualize the biofilm structure, including the distribution of cells inside the extracellular matrix and the areas of surface adhesion. We compared classical SEM (chemically fixed samples) with cryogenic SEM, which employs physical sample preparation based on plunging the sample into various liquid cryogens, as well as high-pressure freezing (HPF). For imaging the biofilm interior, we applied the freeze-fracture technique. In this study, we show that the different means of sample preparation have a fundamental influence on the observed biofilm structure. We complemented the SEM observations with Raman spectroscopic analysis, which allowed us to assess the time-dependent chemical composition changes of the biofilm in vivo. We identified the individual spectral peaks of the biomolecules present in the biofilm and we employed principal component analysis (PCA) to follow the temporal development of the chemical composition.

• Klíčová slova
• Raman spectroscopy, biofilm, cryo-SEM, sample preparation, scanning electron microscopy,
• MeSH
• bakteriální infekce diagnóza   mikrobiologie   MeSH
• biofilmy růst a vývoj   MeSH
• Candida parapsilosis izolace a purifikace   patogenita   ultrastruktura   MeSH
• lidé MeSH
• mikroskopie elektronová rastrovací MeSH
• Ramanova spektroskopie MeSH
• Staphylococcus epidermidis izolace a purifikace   patogenita   ultrastruktura   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články 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.

• Klíčová slova
• Biofilm, Candida, Freeze-fracturing, High-pressure freezing (HPF), Staphylococcus, cryo-SEM,
• 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