Polaron range sputter coater

The indicator strain was grown to mid-log phase (OD₆₀₀ of ~0.6) and incubated with vagococcin T (10× MIC) for 2 h at 37°C with gentle shaking. A culture with no bacteriocin added was used as a control. After incubation, cells were harvested by centrifugation (10,000 × g for 5 min), washed twice in PBS, and resuspended in fixing solution (1.25% [wt/vol] glutaraldehyde, 2% [wt/vol] formaldehyde, PBS) for incubation overnight at 4°C. Fixed cells were then washed three times in PBS and allowed to sediment/attach on poly-l-lysine-coated glass coverslips at 4°C for 1 h. Subsequently, attached cells were dehydrated with an increasing ethanol series (30, 50, 70, 90, and 96% [vol/vol]) for 10 min each and finally washed four times in 100% ethanol. Cells were dried by critical-point drying using a CPD 030 critical-point dryer (Bal-Tec, Los Angeles, CA, USA). Coverslips were sputter coated with palladium-gold using a Polaron Range sputter coater (Quorum Technologies, Lewes, UK). Microscopy was performed on an EVO50 EP scanning electron microscope (Zeiss, Oberkochen, Germany) at 20 kV with a probe current of 15 pA.

The indicator strain was grown to mid-log phase (OD₆₀₀ of ~0.6) and incubated with vagococcin T (10× MIC) for 2 h at 37°C with gentle shaking. A culture with no bacteriocin added was used as a control. After incubation, cells were harvested by centrifugation (10,000 × g for 5 min), washed twice in PBS, and resuspended in fixing solution (1.25% [wt/vol] glutaraldehyde, 2% [wt/vol] formaldehyde, PBS) for incubation overnight at 4°C. Fixed cells were then washed three times in PBS and allowed to sediment/attach on poly-l-lysine-coated glass coverslips at 4°C for 1 h. Subsequently, attached cells were dehydrated with an increasing ethanol series (30, 50, 70, 90, and 96% [vol/vol]) for 10 min each and finally washed four times in 100% ethanol. Cells were dried by critical-point drying using a CPD 030 critical-point dryer (Bal-Tec, Los Angeles, CA, USA). Coverslips were sputter coated with palladium-gold using a Polaron Range sputter coater (Quorum Technologies, Lewes, UK). Microscopy was performed on an EVO50 EP scanning electron microscope (Zeiss, Oberkochen, Germany) at 20 kV with a probe current of 15 pA.