Autosamdri 814

Turkey skin samples (1 inch × 1 inch) inoculated with S. Heidelberg (4 log₁₀ CFU/sq. inch) was dipped in 1% PEO for 5 min at 4°C. Non-inoculated skin samples dipped in sterile DI water or PEOs were used as negative controls. S. Heidelberg dipped in sterile DI water for 5 min served as S. Heidelberg controls. Sample preparation for electron microscopy was conducted as previously described with some modifications (Lee et al., 2014 (link)). Briefly, immediately after each treatment, the skin samples were stored in primary fixative [3% paraformaldehyde, 1.5% glutaraldehyde, and 2.5% sucrose in 0.1 M sodium cacodylate buffer with 5mM calcium chloride and 5mM magnesium chloride (pH 7.4)] for 12 h. Then the samples were fixed in 2% osmium tetroxide and 0.1 M sodium cacodylate buffer for 12 h. Then the samples were washed in ultrapure water (NANOpure Infinity^®; Barnstead/Thermo Fisher Scientific; Waltham, MD, United States) and dehydrated by ascending grades of ethanol series. Samples were processed in a critical point dryer (Autosamdri-814; Tousimis; Rockville, MD, United States) and mounted on aluminum stubs, sputter-coated with gold-palladium, and observed using a scanning electron microscope (S3500N; Hitachi High Technologies America, Inc.; Schaumberg, Illinois; University of Minnesota Imaging Center) at an accelerating voltage of 5.00 kV.

Samples were fixed in 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) overnight at 4°C, rinsed in 0.1 M sodium cacodylate buffer, then placed in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer overnight at 4°C. Specimens were rinsed in ultrapure water (NANOpure Infinity; Barnstead/Thermo Fisher Scientific; Waltham, Maryland), dehydrated in an ethanol series, and processed in a critical point dryer (Autosamdri-814; Tousimis; Rockville, Maryland). Material was mounted on double-sided adhesive carbon tabs on aluminum stubs, sputter-coated with gold-palladium, and observed in a scanning electron microscope (S3500N; Hitachi High Technologies America, Inc.; Schaumberg, Illinois) at an accelerating voltage of 10 kV.

The male lice were removed from the Karnovsky fixative and washed in phosphate buffer for 3 h at 22 ± 1 °C. Samples were post-fixed with 2% osmium, washed with distilled water, then dehydrated with ethanol at increasing concentrations (30, 50, 70, 90, and 100%), and dried with CO₂ using an Autosamdri-814 critical point dryer (Tousimis Research, Rockville, MD). The specimens were mounted on aluminum stubs using double-sided carbon tape. Next, the samples were coated with gold–palladium using a sputter coater (Polaron SC7640; Quorum Technologies, Laughton, East Sussex, UK) for 4 min. To improve conduction, a film of silver particles (OW52765459; Agar Scientific, Stansted, Essex, UK) was applied to the genitalia of nine specimens (two with partially extruded genitalia and seven with fully extruded genitalia). One specimen with fully extruded genitalia was not covered with the film. Images of the complete specimens and the specific structures of the genital area were taken. The samples were examined under a Hitachi S4800 scanning electron microscope (Hitachi High-Technologies) at an accelerating voltage of 10 kV and working distances of 8.7–10.2 mm. Images of 1280 × 960 pixel resolution were acquired.

Small fragments (2–5 mm) of B. crini and H. euphorbiae hindguts were dissected. Fragments were fixed by immersion into paraformaldehyde 2% – glutaraldehyde 2.5% for more than 2 h, washed with water and refixed by osmium tetroxide for 20 min, washed and dehydrated in absolute ethanol. These pieces were placed inside microporous capsules (30 μm pore size, available from Ted Pella Inc. product number 4619) immersed in absolute ethanol, following critical point drying in an Autosamdri 814 (Tousimis). Dry samples were then arranged on SEM stubs with silver conducting paint TAAB S269. Pieces were manipulated under a stereomicroscope Leica MZ9.5 with Dumont forceps number 5. Stubs were examined under a scanning electron microscope Hitachi S-4100. Images were edited with Photoshop CS3 (Adobe).

For microstructure analysis, both formulations were histologically stained for haematoxylin-eosin (H&E) and may-grunwald-giemsa (MGG) (Sigma-Aldrich, St. Louis, USA). Briefly, clots were fixed in 10% formalin for 24 h, dehydrated and then embedded in paraffin. The resulting blocks were cut into 6 µm slices and stained for observation under optical microscope (Leica DMLB, Leica Microsystems, Wetzlar, Germany). Additionally, scanning electron microscopy (SEM) was employed to evaluate the ultrastructure of formulations. Samples were fixed with 2.5% glutaraldehyde, fixed with osmium tetroxide (1% OsO₄ in 0.1 M cacodylate) and finally dehydrated through ascending alcohol concentrations. Thereafter, clots were subjected to critical point drying (Autosamdri 814, Tousimis, Rockville, USA), gold sputter coated and imaged using an electron microscope (S-4800, Hitachi, Japan).

The stem trichome images were acquired using the Nikon SMZ18 stereo microscope equipped with the DS Ri2 colour camera and run by the NiS Elements AR software. The total image magnification was 15x. For scanning electron microscopy (SEM), leaf pieces were fixed for at least 1 h under vacuum while submerged in 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate at room temperature, then stored overnight at 4 °C. Samples were rinsed in buffer, postfixed in 1% osmium tetroxide in buffer overnight at 4 °C, rinsed in ultrapure water (NANOpure Infinity®; Barnstead/Thermo Fisher Scientific; Waltham, Maryland), and dehydrated in an ethanol series. Samples were then processed in a critical point dryer (Autosamdri-814; Tousimis; Rockville, Maryland). Material was mounted on aluminum stubs using double-sided adhesive carbon tabs and colloidal graphite, sputter-coated with gold-palladium, and observed in a scanning electron microscope (S3500N; Hitachi High Technologies America, Inc.; Schaumberg, Illinois) at an accelerating voltage of 10 kV.