100s tem

Semen samples were rinsed with PBS and centrifuged for 500 g for 10 minutes (x2). The overlying PBS was removed without disturbing the pellet and the TEM buffer added (2.5% glutaraldehyde, 4.0% formaldehyde, 0.02% picric acid in 0.1 mol/l sodium cacodylate buffer) at room temperature for 15 minutes and overnight at 4°C. They were then washed in buffer, exposed to 2% osmium tetroxide (Electron Microscopy Sciences, USA) for 1 hour, dehydrated through increasing concentrations of ethanol up to 100%, and embedded in Spurr's resin (Electron Microscopy Sciences, USA). Thick plastic sections were cut and stained with toluidine blue in borate buffer. When spermatozoa were detected, ultrathin sections were cut, stained with uranyl acetate and lead citrate, and studied under a JEOL 100S TEM. Men with recurrent analyses of their specimen at our laboratory were grouped, and concentration plotted and indexed on their round cell episode(s). In order to identify an eventual temporal distribution, we plotted the round cell manifestation for each month to identify any seasonal distribution.

X-ray diffraction data were acquired with a Rigaku/Dmax2100 equipment equipped with Cu radiation (K_α1 = 1.5406 Å). Infrared spectra were measured with a Spectrum GX system/Perkin Elmer using a resolution of 4 cm⁻¹ and averaging over 64 scans. Spectra from the cuticle of C. Chrysargyrea and crab chitin were measured in attenuated total reflection mode and for uric acid the KBr pellet technique was employed. For transmission electron microscopy the sample was first fixed in 3% glutaraldehyde for 2 h, then rinsed in sodium cacodylate buffer, immersed in osmium tetroxide for 1 h, rinsed in deionized water, immersed in 1% uranyl acetate for 1 h, rinsed again in deionized water, followed by a dehydration series starting with 30% ethanol and ending with 100% ethanol, and embedding in a resin. The sample was imaged in JEOL 100 S TEM instrument.

Transmission electron microscopy analysis was used both to assess the morphology of the two different endosymbionts in E. aediculatus, and to verify the potential presence of Rheineimera sp. EpRS3 inside E. aediculatus EASCc1 cells which underwent culture experiments. For TEM analysis, eukaryotic cells sampled from each of the protist sub-cultures of culture experiments were fixed in a 1:1 mixture of 2% OsO₄ in sterile distilled water and 2.5% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.4); then cells were ethanol-dehydrated, transferred to 100% acetone, and embedded in an Epon araldite mixture (Modeo et al., 2013a (link)).
After overnight growth in liquid TSB medium the pellet of Rheinheimera sp. EpRS3 (about 10¹⁰ bacterial cells/ml) was fixed for TEM analysis according to Nitla et al. (2018) (link). Prokaryotic cells were fixed in 1.5-ml Eppendorf tubes and all the solutions (fixatives, ethanol, acetone, and Epon araldite embedding mixture) were directly added. At each step, the Eppendorf tube with prokaryotic cells was vortexed and centrifuged for 5 min at 5,500 × g; then, after discharging the supernatant, the next solution was added, and the pellet was re-suspended.
Ultrathin sections of both eukaryotic and prokaryotic cell TEM preparations were placed on copper grids and stained with uranyl acetate and lead citrate prior to observation with a JEOL 100S TEM.