K850 critical point dryer

Images of mature leaves were taken with a Nikon SM225 Stereo microscope (Japan). To show the micromorphological traits, a scanning electron microscope (SEM) was used. The mature leaves were fixed in Formaldehyde-acetic acid-ethanol (FAA) (methanol: acetic acid: ethanol: water = 10:5:50:35), cut into small pieces, and washed in 70% alcohol. Then, they were dehydrated in an increasing alcohol series and iso-amyl acetate series. Afterward, the material was critical-point dried using liquid CO₂ with a K850 critical-point dryer (Quorum). The leaf pieces were then mounted on aluminum stubs and sputter-coated with gold using a JS-1600 sputter coater (HTCY). Photos were taken with a Hitachi S-3400 SEM (Hitachi, Tokyo, Japan).

SEM images were acquired by a JSM-7800F (JEOL, Akishima, Japan) A field emission scanning electron microscope; puffed rice were dried with a K850 critical point dryer (Quorum Technologies Ltd., Lewes, UK) and coated with a Q150T ES Plus (Quorum Technologies Ltd.) at 18 mA for 90 s with 2 nm AuPd.

E. coli K-12 ORN172 bacteria harboring pN3 were grown on LB agar plates supplemented with tetracycline and kanamycin. The next day, bacteria were gently scraped off using a bacterial inoculation loop and lightly resuspended in 500 μL saline. 100 μL of the bacterial suspension were applied onto fibronectin-coated 13 mm round “Deckglaser” coverslips and incubated for 1 h at room temperature. Following the incubation, coverslips were transferred into 24-well plates, 1 coverslip per well, filled with 1 mL fixation buffer containing 80 mM PIPES NaOH, pH 6.8, 1 mM MgCl₂, 150 mM sucrose, 2% paraformaldehyde (Electron Microscopy Sciences, [EMS]), 2% glutaraldehyde (EMS) for 20 min at room temperature. Samples were then washed twice for 2 min with 1 mL of 0.1 M sodium cacodylate buffer pH 7.4 (EMS). Secondary fixation was performed with 2% osmium tetroxide in 0.1 M sodium cacodylate for 10 min. The samples were washed twice for 2 min each in double-distilled water (DDW) and then dehydrated (2 min, twice for each step) under a series of ethanol concentrations (7.5%, 15%, 30%, 50%, 70%, 90%, 95%, and 100%). Next, samples underwent critical-point drying using a K850 critical-point dryer (Quorum Technologies). Coating was performed with ~1 nm of iridium using a Q150T coater (Quorum Technologies). Samples were imaged in a Merlin scanning electron microscope (Zeiss).

For observations of the epidermis surface, small pieces of S. officinalis leaf and stem were fixed in standard solution of 25mM phosphate buffer (pH = 7) with 3% glutaraldehyde (Sigma Aldrich, Munich, Germany), and 0.01% Tween 20. The samples were kept overnight in the same fixative solution without Tween 20. Another fixative used with the same procedure was FAA fixative (50% ethanol, 35% distilled water, 10% formaldehyde, and 5% glacial acetic acid). After overnight fixation at 4 °C samples were washed in phosphate buffer three times at 10-min intervals. The fixed plant material was dehydrated in graded ethanol series (30%, 50%, 70%, and 95%) and immersed in absolute ethanol (Sigma Aldrich, Munich, Germany). Ethanol was evaporated and fixed samples were further dried using critical-point dried in liquid CO₂ using a K 850 Critical Point Dryer and sputter-coated with gold (10 mm thickness) using Q1 50R ES (Quorum, Lewes, UK). The observations were carried out using a FE–SEM Tescan Mira III (Tescan, Brno, Czech Republic) at an accelerating voltage of 4 kV [51 (link),52 (link)].

Scanning electron microscopy analysis of the leaves was carried out for two-month-old WT and STO-OE lines according to the previous procedure, with minor modifications [72 (link)]. The leaves were fixed in 2.5% glutaraldehyde at 4 °C. The tissue blocks were washed with 0.1 M Phosphate Buffer (PB, pH 7.4) three times and transferred to 1% osmium tetroxide (OsO4) made in 0.1 M PB (pH 7.4) for 1–2 h at room temperature. Subsequently, the tissue blocks were washed three times using 0.1 M PB (pH 7.4). The washed tissue blocks were dehydrated in a graded ethanol series and immersed in tert-butanol for 30 min. The samples were dried in the K850 Critical Point Dryer (Quorum Technologies Ltd., Lewes, UK), and then, attached to metallic stubs using carbon stickers and sputter-coated with gold for 30 s. The images were observed and taken with a scanning electron microscope. Stomatal density and guard cell length and width were determined from the images. A stomatal index (SI) was calculated using the following formula: SI = (number of stomata) /(number of stomata + number of other epidermal cells) × 100.

Bacteria were cultivated in small-scale under conditions described in Table 1. A volume corresponding approximately to 2 × 10⁹ CFU was pelleted (6000 × g, 15 min, 4°C) and washed twice in 100 mM sodium cacodylate/5 g/L NaCl, pH 7.2. Bacteria were then fixed by 3% glutaraldehyde in the same buffer at RT for 1 h and then at 4°C overnight with slow rotation. Sterility of the fixed solution was checked by aliquot plating. The washed bacterial cells were then allowed to sediment overnight onto poly-L-lysine treated circular coverslips at 4°C. The coverslips with attached bacteria were post-fixed with 1% OsO₄ for 1 h at room temperature and three times washed with ddH₂O. Washed coverslips with the bacteria were dehydrated through an alcohol series (25, 50, 75, 90, 96, 100, and 100%) followed by absolute acetone and critical point dried from liquid CO₂ in a K850 Critical Point Dryer (Quorum Technologies Ltd.). The dried samples were sputter coated with 3 nm of platinum in a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies Ltd.). The final samples were examined in a FEI Nova NanoSEM 450 scanning electron microscope (Thermo Fisher Scientific) at 5 kV using CBS and TLD detectors.

Streptomyces spores were fixed with 3% glutaraldehyde overnight at 4°C. The fixed spores were extensively washed and then allowed to sediment at 4°C overnight onto circular coverslips treated with poly-L-lysine. The coverslips with attached spores were dehydrated through an alcohol series followed by absolute acetone and critical point dried from liquid CO₂ in a K850 Critical Point Dryer (Quorum Technologies Ltd, Ringmer, UK). The dried samples were sputter-coated with 3 nm of platinum in a Q150T Turbo-Pumped Sputter Coater (Quorum Technologies Ltd, Ringmer, UK). The final samples were examined in a FEI Nova NanoSEM scanning electron microscope (FEI, Brno, Czech Republic) at 5 kV using CBS and TLD detectors. Beam deceleration mode of scanning electron microscope was used in some cases for minimization of charging artifacts.