Preparation of E. coli culture for electron microscopy

Escherichia coli was grown in TSA for 18 h after which a single colony was inoculated into Tryptic Soy Broth aseptically and incubated at 37 °C on a shaker for 18 h. After this, an inoculum equivalent to a McFarland No 1 standard (3.6 × 10⁸ cfu/mL) was prepared from the 18 h culture. Prior to this, TSA was prepared, and 5 mL of the molten agar was gently poured into 35 mm diameter sterile tissue culture plates to form a smooth and evenly spread surface and allowed to solidify in a sterile environment. The 35 mm agar plates were inoculated with the appropriately adjusted E. coli suspension which were spread evenly on the agar surface with a sterile glass spreader. The plates were then incubated at 37 °C for 12 h under aerobic conditions. The plates were divided into two groups. Plates in group one was flooded with 100 μl of E. zeyheri extract while those in group two were flooded with the extract of S. legatii, both at 0.04 mg/mL in acetone. This concentration represented the minimum inhibitory concentration of the extracts on the test bacterium in our previous study. One plate was flooded with 100 μl of 50% acetone to represent the solvent control. One plate also served as the untreated control, while another was treated with 100 μl gentamicin (1 μg/mL) as positive control. The plates were then incubated under aerobic conditions at 37 °C. After 0, 3, 6, 12, and 24 h, separate plates were removed from the incubator and flooded with 1 mL of 2.5% glutaraldehyde in 0.075 M phosphate buffer solution (pH 7.4) to fix the samples for 60 min. The bacterial biofilms were then collected from each plate using sterile loops and transferred into 2 mL microcentrifuge tubes containing 1.5 mL of 0.5% glutaraldehyde in order to fix the cells for 1 h. Glutaraldehyde was removed with a pipette and the cells were washed thrice with 0.075 M sodium buffer for 10 min each. The samples were then fixed with osmium tetroxide (OsO₄, Merck, Darmstadt, Germany) in a fume hood for 30 min. Osmium tetroxide was removed and the cells were rinsed three times with the sodium phosphate buffer. Dehydration was done with increasing ethanol concentrations of 50, 70, 90 and 100% for 15 min each. The 100% ethanol step was repeated three times before preparation for scanning and transmission electron microscopy.