SEM/STEM Observation of Biofilm/Mineral Interface 1161
Figure 1. Device developed for the growth of biofilms as flocs [in polyvinyl chloride (PVC) tube] or attached on a surface (inner face of PVC tube or sand surface by sampling in the column).
Briefly, flocs were incubated in the dark with a DAPI solu- tion (5 µg/L) for 15min. Samples were then washed with phosphate-buffered saline (PBS) 1× (NaCl 0.8 g/L, KCl 0.2 g/L, Na2HPO4 1.44 g/L, KH2PO4 0.24 g/L) and labeled with lectin PNA-FITC [Peanut Aglutinin, lectin from Arachis hypogaea (peanut) FITC conjugates; Sigma-Aldrich] or ConA-FITC [lectin from Canavalia ensiformis FITC conjugate; Sigma- Aldrich, St. Louis,MO, USA] (both lectins at 50µg/mL in PBS 1× buffer) in the dark for 20 min. Samples were washed before observation in a fluorescentmicroscope (Zeiss Axio Imager Z1, Plan Neofluar ×100 objective (immersion); AxioVision 4.6 software). To avoid out of focus images and to decrease back- ground fluorescence, a z-stack approach for fluorescent labeling imaging of the flocswas applied. Using this approach, 15 images of the same area in the biofilm were taken at different focus/ depths over an overall distance of 0.3µm, and then merged.
Lectin–gold labeling and sample preparation for observation for electron microscopy Labeling with lectins coupled to gold particles (15 and 40nm) (PNA and ConA; EY Laboratories, San Mateo, CA, USA—ready-to-use lectin solutions at a concentration of 10–20 µg/mL) was applied to the samples before SEM observations. Labeling was performed on the fresh biofilm immediately after sampling. Biofilms were washed once using PBS 1× buffer. They were then covered with the ready- to-use solutions of lectin coupled to gold clusters for 90 min in the dark. After removing the lectin–gold solution, biofilms were washed three times with PBS 1× buffer. For STEM-in- SEM and (S)TEM (in TEM), labeling was performed before glutaraldehyde fixation. For STEM-in-SEMand (S)TEM (in TEM) observations,
samples (lectin–gold labeled biofilms) were washed three times with cacodylate buffer (0.1M, pH7.2) and submitted to a prefixation step. For this, cells were fixed in 2.5% glutar- aldehyde in Cacodylate buffer 0.15Mat pH 7.2 for 2h, under
vacuum. Samples were then postfixed in 1% osmium tetr- oxide for 1 h and extensively rinsed before inclusion in 2% agar gel (bacteriological quality) at 60°C. Agar was used to stabilize biofilm samples. Stabilizing samples in gel make them easy to cut into small pieces of 1mm3 before dehydra- tion and inclusion. Samples were dehydrated through a series of graded ethanol solutions from 70 to 100%. The specimens were infiltrated with a mixture of acetone–Epon (50/50) for 3 h then with pure Epon for 16 h. Finally, the specimens were embedded in DMP30-Epon for 24h at 60°C. Ultrathin sec- tions (80nm) were obtained on a Leica UC6 microtome (Wetzlar, Germany), collected on copper hexagonal grids. For STEM-in-SEM observations, samples (biofilm
sections on copper grids) were coated with 2 nm Carbon in a Cressington 208C (Cressington, Watford, UK) or 0.5nm Pt/Pd in a Cressington 208HR. Indeed, noncoated section on grids did not withstand the electron beam energy and serious degradation was observed when increasing magnification.
Cryo-SEM
Twodifferent cryo-SEMoptions were used. The first option is the use of a high-vacuum Hitachi S4500 (Tokyo, Japan) cold- FEG SEMequipped with a Quorum Technology Polaron LT 7400 (Lewes, UK) cryo-SEM preparation system (including cryo-fracturation, gold coating). The samples were frozen in nitrogen slush at −210°C, and then transferred to the pre- paration chamber. No coating was applied on the samples. Cryo-fractionation (if necessary) was processed in the pre- paration chamber at −180°C. Cryo-fractionation allows observation of the internal organization (including the EPS network) of the biofilm. Then, SEM observations were per- formed at −108°C at low voltage (1 kV) using a secondary electron (SE) detector. A preobservation step at −70°C was applied in the SEM chamber to remove the ice layer formed on the surface of the sample during its transfer.
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