Jsm 6500f field emission scanning electron microscope

Scanning electron microscopy (SEM) was performed to verify the attachment of cells and visualize the formation of a biofilm extracellular matrix. For each experimental replication, three additional coupons that were inoculated with S. Reading outbreak strain 0330 were incubated in static and fluidic shear stress conditions and prepared for SEM. The coupons were removed from their respective incubation conditions with sterile forceps and gently rinsed with sterile deionized water to remove loosely attached planktonic cells. The coupons were placed in a new 24-well plate and 1 mL of fixative (2.5 glutaraldehyde, 2% paraformaldehyde, 0.1 M sodium cacodylate) was pipetted into each well. The coupons were incubated at room temperature (include temperature here) for 45 min in the fixative and then placed in a new sterile 24-well plate to dry. Each coupon was then sputter coated with 30 µg of platinum and imaged using a JEOL JSM-6500F Field Emission Scanning Electron Microscope.

To perform SEM on intact salivary glands within the inner cavity of O. turicata, the cuticle and midgut were removed, discarded, and the tick was washed with PBS, and placed in Karnovsky's fixative. Individual salivary glands were also excised and placed in Karnovsky's fixative. Samples were treated with 2% Osmium tetraoxide for 2 hours then dehydrated by immersion in increasing concentrations of ethyl alcohol, with the final concentration of 100% ethyl alcohol. Cryofracturing was performed on excised salivary glands in liquid nitrogen. Samples were dried using a E300 Critical Point Dryer (Polaron Equipment. Watford, United Kingdom) and viewed under a JEOL JSM-6500F Field Emission scanning electron microscope (JEOL USA, Inc., Peabody, MA, USA).

Triplicate decellularized plugs were fixed in 10% neutral buffered formalin, decalcified, and embedded in paraffin. Sections were stained with hematoxylin and eosin to demonstrate the extent of decellularization. Additional plugs were placed in Karnovsky fixative, washed in PBS, and dehydrated through graded alcohols. They were then diametrically cryofractured and further dried using ethanol and hexamethyldisilazane. After the final air drying, the samples were imaged in a JSM-6500F Field Emission Scanning Electron Microscope (JEOL Ltd., Peabody, MA, USA)).

The samples were fixed with OsO₄ as adapted from [48 (link)]. Samples were placed on 0.05-micron mixed-cellulose-esters’ filters (Sterlitech, Auburn, AL, USA) and incubated in 39.3 mM double distilled water solution of OsO₄ for 2 h. Then they were washed 3 times with distilled water (10 min each), dehydrated in graded series of ethanol (30%, 50%, 70%, 80%, 90%) and absolute ethanol, each step 10 min. Absolute ethanol was replaced twice. Then they were washed in hexamethyldisilazane (mixed with absolute ethanol; 30% and 50%) and in absolute hexamethyldisilazane, each step 10 min. The samples were left to dry in air overnight. For examination under JSM-6500F Field Emission Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan), the samples were sputtered with Au/Pd (PECS Gatan 682). Erythrocytes in Figure 2 were imaged as described in [49 (link)].

Isolates were fixed overnight at 4 °C in Karnovsky’s fixative with modification composed of 2.5% glutaraldehyde, 0.4% formaldehyde, and phosphate buffer saline (PBS) at pH 7.4 (137 mM NaCl, 2.68 mM KCl, 10.14 mM Na₂HPO₄, and 1.84 mM KH₂PO₄), and post-fixed the following day with OsO₄, according to the protocol adopted from Lešer et al. 2007 [35 (link)]. Fixatives were removed in three rinsing steps using PBS (10 min incubation in each step). Then, samples were incubated in 2% OsO₄ for one hour, rinsed three times with distilled water (10 min incubation time in each step), and with saturated water solution of thiocarbohydrazide (15 min incubation time), rinsed three times with distilled water (10 min incubation time in each step), incubated again in 2% OsO₄ for 1 h, washed three times with distilled water (10 min incubation time in each step), and dehydrated in graded series of ethanol (30–100%, 10 min in each solution). Absolute ethanol was replaced three times, treated by the graded series of hexamethyldisilazane (mixed 30% and 50% with absolute ethanol and pure, 10 min incubation time in each step), and finally air dried overnight. Fixed and dehydrated samples were Au/Pd coated by Precision Etching and Coating System or PECS (Gatan Inc 682, Pleasanton, CA, USA), and examined using a JSM-6500F Field Emission Scanning Electron Microscope (JEOL Ltd., Tokyo, Japan).

For scanning electron microscopy, specimens were initially prepared by fixing in 2.5% glutaraldehyde in 0.15 M sodium phosphate buffer, pH 7.4, at 4 °C for 24–48 h. Post-fixation was carried out in 1% buffered osmium tetroxide overnight at 4 °C, then samples were dehydrated through a graded ethanol series, followed by critical point drying using a BioRad E3000 critical point dryer (Quorum Technologies, East Sussex, England), or by dehydration with acetone and hexamethyldisilazane. All samples prepared for SEM were sputter coated with 10 nm gold using a Hummer VII (Anatech USA, Union City, California, USA). Micrographs were taken using a JEOL JSM-6500F Field Emission Scanning Electron Microscope at the Central Instrument Facility, Imaging Laboratory, located at Colorado State University. All images were captured digitally as tiff files, and graphically edited to input black image backgrounds (Adobe Photoshop, CS6 13.0.1). All measurements were taken at the widest or longest point of a given structure (Table 1), and are given in μm. Ranges are presented, followed by the means in parentheses.