Spurr low viscosity resin

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. Bacteria were grown to mid-logarithmic phase and washed in 0.1 M cacodylate buffer, pH 7.3 (Canemco & Marivac, Canton de Gore, QC, Canada). The CPS was stabilized using specific antibodies as previously described (46 (link)). Anti-S. suis serotype 2 rabbit serum, produced as previously described (47 (link)), was used to gently resuspend bacteria. Next, cells were immobilized in 4% (w/v) agar in 0.1 M cacodylate buffer, pH 7.3. Pre-fixation was performed by adding 0.1 M cacodylate buffer, pH 7.3, containing 0.5% (v/v) glutaraldehyde, and 0.15% (w/v) ruthenium red for 30 min. Fixation was performed for 2 h at room temperature with 0.1 M cacodylate buffer, pH 7.3, containing 5% (v/v) glutaraldehyde, and 0.05% (w/v) ruthenium red. Post-fixation was carried out with 2% (v/v) osmium tetroxide in water overnight at 4°C. Samples were washed with water every 20 min for 2 h to remove osmium tetroxide and dehydrated in an increasing graded series of acetone. Specimens were then washed twice in propylene oxide and embedded in Spurr low-viscosity resin (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections were post-stained with uranyl acetate and lead citrate and examined using a transmission electron microscope at 80 kV (Hitachi model HT7770, Chiyoda, Tokyo, Japan).

Unless otherwise indicated, chemicals were purchased from Sigma-Aldrich. Transmission electron microscopy was carried out as previously described [35 , 44 (link)]. Briefly, bacteria were grown to mid-logarithmic phase and washed in 0.1 M cacodylate buffer pH 7.3 (Canemco & Marivac, Canton de Gore, QC) containing 2.5% glutaraldehyde and 0.05% ruthenium red. Ferritin was added to a final concentration of 1 mg/mL and incubated for 30 min at room temperature. Cells were then immobilized in 3% agar in 0.1M cacodylate buffer pH 7.3, washed five times in cacodylate buffer containing 0.05% ruthenium red, and fixed in 2% osmium tetroxide for 2 h at room temperature. Afterwards, samples were washed with water every 20 min for 2 h to remove osmium tetroxide and dehydrated in an increasing graded series of acetone. Specimens were then washed twice in propylene oxide and embedded in Spurr low-viscosity resin (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections were post-stained with uranyl acetate and lead citrate and examined using a transmission electron microscope at 80 kV (Hitachi model HT7770, Chiyoda, Tokyo, Japan).

TEM was carried out to confirm CPS expression at the bacterial surface of different mutant strains as previously described (Roy et al., 2015 (link)). Briefly, bacteria were grown to mid-logarithmic phase and resuspended in 0.1 M cacodylate buffer pH 7.3 containing 2.5% (v/v) glutaraldehyde and 0.05% (w/v) ruthenium red. Fixation was performed for 2 h at room temperature. Ferritin (Electron Microscopy Sciences, Hatfield, PA) was then added to a final concentration of 1 mg/ml and incubated for 30 min at room temperature. Afterwards, cells were immobilized in 4% (w/v) agar in 0.1 M cacodylate buffer pH 7.3 and post-fixed with 2% (v/v) osmium tetroxide in water overnight at 4°C. Samples were washed with water every 20 min for 2 h to remove osmium tetroxide and dehydrated in an increasing graded series of acetone. Specimens were then washed twice in propylene oxide and embedded in Spurr low-viscosity resin (Electron Microscopy Sciences). Thin sections were post-stained with uranyl acetate and lead citrate and examined with a transmission electron microscope at 80 kV (model JEM 1230, Jeol, Tokyo, Japan).