Sigma vp40 field emission scanning electron microscope

For scanning electron microscopy (SEM), glass discs were precleaned with acetone, ethanol, and water before sterilization. Biofilms were grown as stated above and rinsed by water before being fixed by 2.5% phosphate‐buffered glutaraldehyde for 30 min at 4 °C as previously described 25. The fixed samples were dehydrated in a graded series of cold ethanol/water mixture (increasing from 10%, 20%, 30%, 50%, 70%, 80%, 90%, 95% to 100% ethanol) for 10 min each. Critical point dryer (Leica EM CPD300; Leica Microsystems, Buffalo Grove, IL, USA) was used to dehydrate the samples after which the samples were coated with a gold/palladium target using a high vacuum sputter coater (Leica ACE600; Leica Microsystems). The biofilm discs were observed with a SIGMA VP40 field emission scanning electron microscope (Carl Zeiss, Inc. Oberkochen, Germany) in high vacuum mode at 2 kV. Results presented are representative of at least three independent experiments. Cell diameters in SEM images were measured via imagej (NIH, Bethesda, MD, USA). For each condition, a total of 150 cells were measured across three separate experiments. Data presented as mean ± SEM. Distributions were created using bins of 0.2 μm. Coefficient of variance (CV) was calculated using the formula: $\text{CV}(\%)=\text{standard deviation}(\mathrm{\sigma })/\text{mean}(\mathrm{\mu })\ast 100$

Both the surface and tissue cross-section of the burn eschar were evaluated by scanning electron microscopy (SEM). To visualize the surface of the wound, seven millimeter biopsy punch samples were fixed with 2.5% phosphate-buffered glutaraldehyde for at least 24 hours at 4 °C. Fixed specimens were processed through a graded series dehydration using cold ethanol/water (10%, 30%, 50%, 70%, 80%, 90%, 95%, 100%) and an incubation time of 10 minutes followed by critical point drying (EM CPD300, Leica Biosystems Inc. Buffalo Grove, IL). Immediately prior to examination, the wound biopsy punch samples were coated with carbon and gold/palladium (Leica ACE600 Coater). The histological cross-sections were deparaffinized with 100% xylene and air dried prior to coating with carbon and gold/palladium. Both sets of specimens (biopsy punches and tissue cross-sections) were visualized using a Sigma VP40 field emission scanning electron microscope (Carl Zeiss, Inc. Germany) in high vacuum mode at 2 kV.