S 3000n scanning electron microscope

In addition to measuring the effects of CDB against S. aureus, SEM observation was performed, with some minor modifications (Jiamboonsri et al., 2011 (link); Singh et al., 2017 (link)). Briefly, the prepared inoculum (300 μL) of JP-2541 and S. aureus ATCC 29213 was transferred into LB broth (2.7 mL) in the presence and absence of 1 × MIC CDB, the positively charged glass slide was placed into each well and subsequently incubated at 37°C overnight. The bacterial cells on the coverslip were fixed in 2.5% (w/v) of glutaraldehyde at 4°C for 4 h and rinsed with 0.1 M of phosphate buffer (pH 7.2), followed by dehydration in graded ethanol (30, 70, and 100%) and drying at the room temperature for overnight. Finally, the dried samples were covered with gold and observed under the S-3000N scanning electron microscope (SEM) (Hitachi, Japan) at various levels of magnification.

Sample preparation: The thoracic cavities of the rabbits and the mice were cut open by using scissors and the hearts were removed. After irrigating with phosphate buffered saline (PBS), the hearts were fixed in a solution of 2.5% glutaraldehyde (Res Group Co., Ltd. chemical reagents) for 2 h. Then the atria and ventricles were cut open, and all of the heart valves (including aortic, mitral and tricuspid) were removed. The valves were placed in a freeze-dryer (ES-2030 vacuum freeze-drying device, Hitachi Japan) to be frozen, dehydrated, and dried with tert-butanol for 2–3 h at a temperature of −10 °C.
Characterization of the microstructure on the surface of the heart valve: The heart valves were observed by using the scanning electron microscope (S-3000N scanning electron microscope SEM, Hitachi Japan) at a voltage of 10 kV in order to characterize the microstructures on their surfaces. To improve the conductivity of the sample, before being observed the dried heart valves were treated with spray-gold (E-1010 ion sputtering device, Hitachi Japan) having a coating thickness of approximately 5 nm.

Cells cultured on coverslips were fixed with 2.5% glutaraldehyde overnight at 4 °C, followed by washing in phosphate-buffered saline (PBS, pH = 7.4) and dehydration. Hitachi S-3000N Scanning Electron Microscope (SEM; Hitachi, Tokyo, Japan) was used to examine the cells and images were taken at the magnification of ×2000.

To observe pollen-pistil interactions in the various crosses, pollen grains with the highest germinability were collected from flowers and used to artificially pollinate the most receptive stigmas. Pollen germination on stigmas was investigated using a slightly modified published procedure [20 ]. Samples were collected at 1, 2, 4, 8, 12 and 24 h after pollination (HAP). Each sample included 20 pistils, of which 10 were immediately fixed in FAA solution and observed using an Axioskop 40 fluorescence microscope. The pollen grains and tubes were analyzed according to the aniline blue staining method. The other 10 pistils were immediately fixed in 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) and analyzed by an S-3000N scanning electron microscope (SEM) (Hitachi, Tokyo, Japan). The fixed SEM samples were dehydrated in an ethanol series (40, 70, 90 and 100%) for 15 min at each concentration, and then subjected to critical point drying before being coated with gold and analyzed using SEM [20 ]. The same number of self-pollinated pistils (i.e., SP × SP, DP × DP, and MP × MP) was treated as described above as control samples. Each experiment was repeated three times.