Critical point dryer

For the scanning electron microscope (SEM) imaging, the tissue sections were collected onto the cover slips (32 × 24 mm). The sections were then de-waxed twice in xylene for 10 min, then three times in 100% ethanol for 10 min each followed by two immersions in acetone for 10 min. Each slide was then critical point dried in liquid CO₂ in a critical point dryer (Hitachi, Tokyo, Japan) and ion coated (IB-2, Eiko, Tokyo, Japan) before documentation in the SEM (Inspect S, FEI, Hillsboro, OR, USA).

hFOB 1.19 cells were seeded at 5×10⁴ cells/ml on surface of disc samples, and incubated for 3 days at 33.5°C in serum-containing media. Discs incubated without cells were as negative controls. Cells growing on glass coverslips were used as controls for cell growth. Samples were washed carefully with PBS and fixed overnight in 4% glutaraldehyde buffered in PBS at 4°C. After washing thrice in PBS, the samples were dehydrated in a graded series of ethanol solutions (10%- 100%) for 10 min each, followed by drying in a Critical Point Dryer (Hitachi, Tokyo, Japan). Samples were mounted on stubs and sputtered with gold using an Ion Coater (Eiko, Tokyo, Japan), and then visualized using a SEM at 15 kV to 20 kV.

SEM was used to observe biofilm formation and effects of antimicrobial drugs on biofilm formation12 (link). In order to observe the morphological changes associated with biofilms following moxifloxacin treatment, biofilms were allowed to grow on sterile flat-bottom 6-well polystyrene tissue culture plates (LabServ, Thermo Fisher Scientific, USA) in the presence or absence of moxifloxacin treatment. Fresh Cation-Adjusted Mueller Hinton Broth (CAMHB) without any antibiotic was used as a control. The samples were fixed with 2.5% glutaraldehyde for at least 4 h at 4 °C. After washing with phosphate buffered saline (PBS), the samples were dehydrated in a series of ethanol solutions of increasing concentration (50 to 100%). The cells were subsequently washed with pure acetone, followed by isoamyl acetate. They were then dried with a critical point dryer (Hitachi, Japan). The dried samples were coated with 15-nm gold using an argon automatic sputter coater (Hitachi, Japan). After processing, the samples were viewed with a Philips XL30CP scanning electron microscope.

W. anomalus cells were cultured with a YEPD medium at 28 °C for 8 h. Different concentrations of ethanol (3%, 6%, 9%, and 12%, v/v) were added, and the cells were cultured for 6 h to implement ethanol stress. Cells cultured without ethanol (0% Ethanol) were used as the control for this experiment and other experiments in this study. Yeast cells were centrifuged at 4, 000 × g for 10 min and washed three times with physiological saline. Then, the cells were re-suspended in 2.5% glutaraldehyde for 4 h at 4 °C and washed three times with 0.1 M PBS (pH 7.4). Subsequently, the cells were eluted with a gradient concentration of ethanol solutions (0%, 50%, 70%, 85%, 95%, and 100%) for 15 min each. Yeast cells were dried with a critical point dryer (Hitachi, Japan), coated with a gold/palladium alloy, and observed with an FEI Quanta FEG 450 scanning electron microscopy system (FEI, USA).

To observe microstructure, the bone marrow tissues were taken by the aforementioned method from each group of rats (n=10) on days 0 and 7. The tissues were first fixed with 4% glutaraldehyde (Shanghai Yuanye Biotechnology Co. Ltd, Shanghai, China) for more than 4 h, then were washed three times with the PBS, post-fixed with 1% potassium phosphate-buffered osmium tetroxide (pH = 7.0, provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) for 1 h, and washed three times with PBS. Then, the tissues were dehydrated using a graded series of ethanol (30, 50, 70, 80, 90, 95, and 100%) for 15–20 min at each step, transferred to the mixture of alcohol and isoamyl acetate (provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) for approximately 30 min, and then transferred to pure isoamyl acetate for approximately 1 h. Finally, the tissues were dehydrated in a critical point dryer (Hitachi, Japan) with liquid CO₂. The dehydrated tissues were coated with gold-palladium (provided by Electron Microscope Testing Lab, Chongqing Medical University, Chongqing, China) and observed under a scanning electron microscope (SEM; Hitachi, Japan).

Fresh corneas were first fixed in 2% paraformaldehyde for 24 h, and then in 2.5% glutaraldehyde solution in 0.2 M cacodylate buffer and 1% tannic acid at pH 7.0–7.3 for another 24 h, followed by postfixation with 1% osmium tetroxide solution in 0.2 M cacodylate buffer solution for 1 h. Samples were then dehydrated by a critical point dryer (Hitachi Ltd., Japan) and coated with platinum in an ion sputter coater (Hitachi Ltd., Japan). Finally, the samples were observed and photographed with the scanning electron microscope (Hitachi Ltd., Japan).