Application of lyophilization to prepare the nitrifying bacterial biofilm for imaging with scanning electron microscopy

Abstract

Structure of bacterial biofilms may be investigated using several variants of scanning electron microscopy (SEM). We apply lyophilization to prepare nitrifying bacterial biofilm for conventional SEM imaging in high-vacuum mode (CSEM). We therefore replace standard biofilm fixation in glutaraldehyde cross-linking, ethanol dehydration, and critical-point drying (CPD) with less-invasive low-temperature drying by sublimation in vacuum. We compare this approach with: (1) standard preparation with glutaraldehyde fixation, ethanol dehydration, and CPD before CSEM, (2) cryo-sputter preparation of rapidly frozen biofilm in hydrated state (cryo-SEM), and (3) in situ observation without any sample pretreatment in environmental SEM. Combined imaging with these modalities revealed two distinct immobilization patterns on the polyurethane foam: (1) large irregular aggregates (flocs) of bacterial biofilm that exist as irregular biofilm fragments, rope-like structures, or biofilm layers on the foam surface; (2) biofilm threads adherent to the surface of polyurethane foam. Our results indicate that lyophilization was suitable for preservation of bacterial cells and many forms of structure of extracellular matrix. The lyophilized material could be imaged with high resolution (using CSEM) to generate structural information complementary to that obtained with other SEM techniques.

Controlled communication between physically separated bacterial populations in a microfluidic device

“…Bacterial cells were dehydrated with repeated washing of 2.5% glutaraldehyde and increasing concentrations of ethanol (50%, 70%, 80%, and 90%), after which samples were air dried. Platinum sputtering was used to apply a 5 nm coating to the samples (Emitech K590X/K350) before imaging 40 . Scanning electron microscopy was performed using a Zeiss Sigma VP (Carl Zeiss) with an in-column SE detector and an accelerating voltage of 1 kV.…”

Study on the Structure of Candida Albicans–Staphylococcus Epidermidis Mixed Species Biofilm on Polyvinyl Chloride Biomaterial

“…Despite the fact that BF sample preparation for conventional SEM, including dehydration, fixation, embedding, and stain, can have a certain impact on the structure and components of BF, meticulous examination of the morphology, and spatial distribution of single pathogens in mixed BF can still be accomplished using SEM due its high resolution, exhibiting significantly priority in examining microstructure of mixed BF [15]. Environmental SEM can acquire images of pathogens in living state and avoid damage to the structure of mixed BF, allowing examination of mixed BF in native state.…”

Critical review on biofilm methods

“…For details see Turonova et al (2016) (see also video S1) # Hana Turo nov a. Reuse not permitted. Delatolla et al, 2009;Hannig et al, 2010;Karcz et al, 2012;Pompilio et al, 2015). However, reduced resolution can occur because of the lack of conductivity in the wet sample, or when a rapid image capture is required for samples moving or changing their structure during examination.…”

“…Critical-point drying is the most frequently used method of drying biofilms for SEM, although resulting in a significant loss of EPS (Timp & Matsudaira, 2008). The use of sample lyophilization or hexamethyldisilazane is preferable because it is more conservative (Araujo et al, 2003;Di Bonaventura et al, 2008;Karcz et al, 2012). Finally, since SEM imaging requires a high vacuum, specimens must be solid with negligible outgassing.…”

“…Cryo-SEM also allows for freeze-fracture, where the frozen biological sample is physically broken apart to expose structural detail of the fracture plane, therefore exposing the internal structure of the biofilm that may reveal how the bacteria are interconnected. Deep etching of ultra-rapidly frozen samples permits visualization of the inner structure of cells and their components (Alhede et al, 2012;Karcz et al, 2012). However, Cryo-SEM has some disadvantages: (i) lower image resolution than conventional SEM, as incomplete sublimation of surface moisture may obscure surface details; (ii) melting and cracking of the frozen surface at high magnifications because of the heat generated by the focused electron beam (Alhede et al, 2012); (iii) requirement of highly expensive and specialized equipment, probably explaining its limited use in biofilm studies.…”