Biofilm of S. aureus was grown on glass slides for the light microscopic observation.27 (link) Briefly, sterile round glass sides (ϕ 14 mm) were placed in each well of a 24-well microplate. Then, 500 μL of different concentrations of CHQA in TSB with 1% glucose and 500 μL of logarithmic phase S. aureus inoculum were added to the microplate, and incubated at 37 °C for 24 h. Subsequently, the glass slides were washed thrice with PBS and stained with 0.4% crystal violet. After 5 min, the slides were washed again with distilled water to remove the excess stain and air dried. The stained biofilms were observed with a light microscope (Nikon Eclipse E200, Tokyo, Japan).
The effect of CHQA on the biofilm architecture was further analyzed by a scanning electronic microscope (SEM) with a slightly modified method.3 (link) In brief, the biofilms of S. aureus were developed on glass slides as described above. Then, the glass slides were placed in 24-well microplate, submerged with 2.5% glutaraldehyde at 4 °C for 3 h, and gently rinsed with PBS. Subsequently, the biofilms on the slides were dehydrated in a graded ethanol series of 25%, 50%, 70%, 90% and 100% at 4 °C for 10 min each. After a further critical-point drying, the specimen was sputter-coated with gold and examined under a SEM (JSM-7500, JEOL, Tokyo, Japan).
The effect of CHQA on the biofilm architecture was further analyzed by a scanning electronic microscope (SEM) with a slightly modified method.3 (link) In brief, the biofilms of S. aureus were developed on glass slides as described above. Then, the glass slides were placed in 24-well microplate, submerged with 2.5% glutaraldehyde at 4 °C for 3 h, and gently rinsed with PBS. Subsequently, the biofilms on the slides were dehydrated in a graded ethanol series of 25%, 50%, 70%, 90% and 100% at 4 °C for 10 min each. After a further critical-point drying, the specimen was sputter-coated with gold and examined under a SEM (JSM-7500, JEOL, Tokyo, Japan).