Scanning electron microscope evo ls 15

To characterize particle size/shape, Scanning Electron Microscope EVO LS 15 (Zeiss, Rugby, UK) was used. The powders were scattered onto the surface of aluminum stubs (6 mm height/12.5 mm disc diameter) (Agar scientific, Stansted, UK) using carbon tabs (Agar scientific, UK), followed by a layer of gold coating (15 nm thickness). The samples were measured at four magnifications (30×, 100×, 500× and 1000×).

The cross-section morphology of the cryogenic fracture of the films was obtained via Zeiss Scanning Electron Microscope (EVO LS15), Jena, Germany, operating at a voltage of 5.00 39 kV to 10.00 kV using a thin layer of gold deposited on the samples.

Exponentially growing vegetative cells of BAS WT, BAS ΔprpN and BAS ΔprpN::prpN strains were processed for scanning electron microscopy using the protocol described earlier [41 (link)]. Cells were visualized under Zeiss Scanning Electron Microscope EVO LS15 at 20 KV and analyzed using Smart SEM software.
Ultrastructural details of BAS WT, BAS ΔprpN and BAS ΔprpN::prpN spores were examined using transmission electron microscopy. Samples were processed by two different methods to visualize vegetative sporulating cells population using the protocols described earlier [41 (link), 107 (link)]. Briefly, for pure spore suspension, sporulation medium was removed after sporulation completion and the harvested spores were kept in water for three days to ensure vegetative cell lysis. These were again washed thrice with water followed by resuspension in water to remove vegetative cell debris and fixed for TEM analysis. To visualize intact cells in the sporulation medium, the bacterial cells were harvested and washed thrice with 0.85% saline, resuspended in the same and fixed. Sectioning was done using Leica UC6 ultra-cut and the sections were then observed in FEI Tecnai G2 Spirit at 200 KV.