Analysis of NPs in bacteria by SEM

Both bacterial strains (MDR E. coli and MRSA) were inoculated into 5 mL of sterile LB media in a 50 mL Falcon conical centrifuge tube and incubated at 37°C/200 rpm for 24 hours. The OD was then measured at 600 nm (OD600) using a spectrophotometer. The overnight suspension was diluted to a final bacterial concentration of 10⁶ CFU/mL prior to measuring the OD. A selected 10 µg/mL NP concentration was mixed with LB media and bacterial solution in a six-well plate with a glass coverslip attached to the bottom. The coverslips were pretreated with polylysine to enhance cell adhesion right before the experiment. The plate was placed inside an incubator for 8 hours at 37°C. After the experiment, the coverslips were fixed with a primary fixative solution containing 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer solution for 1 hour. Subsequently, the fixative solution was exchanged for 0.1 M sodium cacodylate buffer and the coverslips were washed three times for 10 minutes. Post-fixation was done using a 1% OsO₄ solution in buffer for 1 hour. Subsequently, the coverslips were washed three times with buffer and dehydration was progressively achieved with 35%, 50%, 70%, 80%, 95% and 100% ethanol – three times for the 100% ethanol. Finally, the coverslips were dried by a liquid CO₂–ethanol exchange in a Samdri-PVT-3D Critical Point dryer. The coverslips were mounted on SEM stubs with carbon adhesive tabs (EMS) after treatment with liquid graphite and then sputter coated with a thin layer of platinum using a Cressington 208HR High Resolution Sputter Coater. Digital images of the treated and untreated bacteria were acquired using an SEM.