Evo 50 ep scanning electron microscope

Algae were treated using the same fixing method described above for TEM. After, coverslips coated with 1 mg mL⁻¹ poly-l-lysine were placed to allow algae to settle for 20 min. The fixed algae culture for SEM examination were washed thoroughly in 0.1 M sodium cacodylate buffer (SCB) and dehydrated with 10 min steps in ascending ethanol series (50–100%) as in Wiik-Nielsen et al. (2016 (link)). The samples were processed in a BAL-TEC Critical Point Dryer CPD 030, (BAL-TEC AG Lichtenstein), and a thin conductive coating of gold–palladium was applied to the samples using a Polar on Sputter Coater SC7640 (Quorum Technologies, UK). The coated samples were mounted on aluminum stubs, examined and photographed with a Zeiss EVO-50-EP scanning electron microscope at an accelerating voltage of 20 kV in the secondary emission mode.

Optical microscopy
(OM) was carried out using a Leica DM LM microscope. Scanning electron
microscopy (SEM) and energy-dispersive spectroscopy (EDS) were performed
using an EVO 50 EP scanning electron microscope (by Zeiss) and an
Inca Energy 200 EDS module (from Oxford Instruments). X-ray diffraction
(XRD) was performed by means of an Xpert MPD setup (by Philips, in
thin film mode and with Cu K_α =
1.5406 Å). Magnetic characterization was carried out using a
MicroMag 3900 (from Princeton Measurement Corp.) vibrating sample
magnetometer (VSM). XRF measurements were performed by means of an
X-RAY XAN apparatus (by Fischerscope). The instrument used to get
atomic force microscopy (AFM) topographical data was a Solver Pro
(by NT-MDT). A CM 10 setup was employed to acquire transmission electron
microscopy (TEM) images.

Overnight cultures were diluted to approximately OD₆₀₀ = 0.1 in BHI. When OD₆₀₀ reached 0.4, the cultures were diluted at 1:250. Antibiotics and IPTG were added when appropriate. The cultures (10 mL) were grown to OD₆₀₀ = 0.3 and 1 volume of fixation solution, containing 5% (wt/vol) glutaraldehyde and 4% (wt/vol) paraformaldehyde in 1×PBS, pH 7.4, was added. The tubes were carefully inverted a few times and incubated for 1 h at room temperature before being placed at 4°C overnight. The following day, the cultures were centrifuged at 5000 × g, and the pellets were washed three times with PBS. Further preparations of samples to be analyzed with TEM were performed as described before (69 (link)).
Samples for SEM were, after washing with PBS, dehydrated with EtOH, essentially in the same manner as for sample preparations for TEM (69 (link)). The samples were subjected to critical point drying by exchanging the EtOH with CO₂. The samples were then coated with a conductive layer of Au-Pd before being analyzed in a Zeiss EVO50 EP Scanning electron microscope. Images were analyzed and prepared using Fiji (71 (link)).

The indicator strain was grown to mid-log phase (OD₆₀₀ of ~0.6) and incubated with vagococcin T (10× MIC) for 2 h at 37°C with gentle shaking. A culture with no bacteriocin added was used as a control. After incubation, cells were harvested by centrifugation (10,000 × g for 5 min), washed twice in PBS, and resuspended in fixing solution (1.25% [wt/vol] glutaraldehyde, 2% [wt/vol] formaldehyde, PBS) for incubation overnight at 4°C. Fixed cells were then washed three times in PBS and allowed to sediment/attach on poly-l-lysine-coated glass coverslips at 4°C for 1 h. Subsequently, attached cells were dehydrated with an increasing ethanol series (30, 50, 70, 90, and 96% [vol/vol]) for 10 min each and finally washed four times in 100% ethanol. Cells were dried by critical-point drying using a CPD 030 critical-point dryer (Bal-Tec, Los Angeles, CA, USA). Coverslips were sputter coated with palladium-gold using a Polaron Range sputter coater (Quorum Technologies, Lewes, UK). Microscopy was performed on an EVO50 EP scanning electron microscope (Zeiss, Oberkochen, Germany) at 20 kV with a probe current of 15 pA.