Es 2030

It is reported that S. pneumoniae in vitro biofilms formed on abiotic surfaces for 10–12 h consist of a three-dimensional structures with significant thickness (25–30 μm) (Moscoso et al., 2006 (link)). In this study, we evaluated the effect of AME/CME on 18-h pre-established in vitro biofilm using scanning electron microscope (SEM). In vitro biofilms of S. pneumoniae D-39 were grown as described above for 18 h and treated with indicated concentration of AME/CME solution for 6 h; the control biofilms were treated with PBS. After incubation, the biofilms were washed, pre-fixed by immersion in 2% glutaraldehyde in 0.1 M phosphate buffer, and post-fixed for 2 h in 1% osmic acid dissolved in PBS. The biofilm samples were then treated with increasing concentrations of ethanol (60–95%), followed by t-butyl alcohol. The samples were dried in a freeze dryer (ES-2030, Hitachi, Tokyo, Japan) and coated with platinum using an ion coater (IB-5, Eiko, Kanagawa, Japan). The images were captured under field emission-SEM (FE-SEM; Hitachi, S-4700, Tokyo, Japan).

SEM using (Hitachi S-4800, Tokyo, Japan) was used for the investigations of cellular alignment and morphological variations. After the cells were cultured on the substrates with and without cisplatin concentrations, the 2 hr incubation using 2.5 vol.% glutaraldehyde (Wako, Japan) in PBS was done for fixation and preservation of the cells morphology. Then, stepwise incubations with several of ethanol concentrations (10, 40, 60, 80, and 100%) for 5 min were used for the cell dehydration, respectively. After that, the freeze drying (Hitachi Es 2030, Tokyo, Japan) was done for desiccation of samples. Finally, the cells over nanopatterns were observed at an acceleration voltage in the range 1–5 kV.

For transmission electron microscope (TEM) observation, first fertilized egg envelopes were pierced a hole with injection needle and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 12 h at 4 °C. After prefixation, the specimens were washed twice in the same buffer solution for 20 min. and then postfixed in 1% osmium tetroxide solution in 0.1 M phosphate buffer solution (pH 7.4) for 2 h at room temperature. Specimens were dehydrated in ethanol, cleared in propylene oxide, and embedded in an Epon mixture. Ultrathin sections of embedded fertilized egg envelope were taken with an ultramicrotome (Ultracut E, Reichert-Jung, Austria) at a thickness of about 60 nm. The ultrathin sections were mounted onto copper grids, double stained with uranyl acetate followed by lead citrate, and observed with a transmission electron microscope (JEM-1400, JEOL, Japan).
For scanning electron microscope observation, prefixation, postfixation and dehydration were conducted by following the same procedure as that for TEM. The samples were replaced with tert-butyl alcohol and freeze dried (ES-2030, Hitachi, Japan). The samples were coated with Pt by ion sputter (E-1045, Hitachi, Japan). Subsequently, the fertilized eggs were observed under the table top scanning electron microscope (TM-1000, Hitachi, Japan).