Libra 120 transmission electron microscope

Cells were stimulated with ionomycin (2 h) or ATP (15 min) in serum-free cell culture medium. For pre-embedding immunogold labelling, cells were fixed in 4% paraformaldehyde (30 min), permeabilised with 0.2% Triton X-100/PBS (10 min, on ice), incubated with YB-1 specific antibody (ab76149, Abcam; Cambridge, UK) in PBS (30 min), and stained with secondary antibody conjugated to 1 nm colloidal gold particles (Nanoprobes; Yaphank, NY, USA) (30 min). After fixation with 2.5% glutaraldehyde (90 min) and silver enhancement with HQ silver enhancement kit (Nanoprobes; Yaphank, NY, USA), cells were fixed again in Karnovsky’s fixative and stored at 4 °C. Cell pellets were embedded in 3.5% agarose at 37 °C, coagulated at room temperature, and fixed again in Karnovsky’s solution. Post-fixed samples (1% OsO₄, 20 min, on ice) were rinsed with distilled water, block-stained with uranyl acetate (2% in distilled water), and dehydrated in ascending series of alcohol dilutions to 100%. Samples were infiltrated with propylene oxide, embedded in glycide ether, and cut using an ultramicrotome (Ultracut; Reichert-Jung/Leica Microsystems; Wetzlar, Germany). Ultrathin sections (30 nm) were mounted on copper grids and analysed using a Zeiss LIBRA 120 transmission electron microscope (Carl Zeiss; Oberkochen, Germany) operating at 120 kV.

S. epidermidis 1457 WT, ∆tagE, ∆pgcA, and ∆gtaB were grown until stationary phase and fixed at an OD₆₀₀ of 10 in 200 µL Karnovsky’s fixative (3% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4) for 24 h. Samples were then centrifuged at 1,400 × g for 5 min, supernatant was discarded, and pellets were resuspended in approximately 20 µL agarose (3.9%) at 37°C, cooled to room temperature, and cut into small pieces. Postfixation was based on 1.0% osmium tetroxide containing 2.5% potassium ferrocyanide (Morphisto) for 2 h. After following the standard methods, samples were embedded in glycide ether and cut using an ultramicrotome (Ultracut E, Reichert). Ultra-thin sections (30 nm) were mounted on copper grids and analyzed using a Zeiss LIBRA 120 transmission electron microscope (Carl Zeiss) operating at 120 kV.

Light microscopy of laboratory-grown Zygnema sp. C was performed using a Zeiss Axiovert 200 M light microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a Zeiss Axiocam MRc5 camera. The Zygnema sp. field samples were observed in an Olympus BX53 microscope with an Olympus DP72 microscope digital camera. Sample preparation for TEM followed the protocol of Holzinger et al. (2009) (link) as described by Rippin et al. (2019) (link). Chemical fixation, ethanol dehydration, and embedding in modified Spurr’s resin (Low viscosity embedding kit, Science Services, Munich, Germany) were conducted immediately after sampling in Svalbard. The embedded material was transferred to the laboratory and ultrathin sections were prepared from the embedded material (Reichert Ultracut, Leica Microsystems, Wien, Austria), counterstained, and investigated with a Zeiss Libra 120 transmission electron microscope (Carl Zeiss AG, Oberkochen Germany) at 80 kV, and images were generated with a 2 k SSCCD camera (Albert Tröndle Restlichtverstärker Systeme, Moorenweis, Germany).