Fei tecnai biotwin spirit microscope

Ts2631 endolysin (final concentration of 7.4 µM) was mixed with 10⁸ of T. thermophilus HB8 or A. baumannii CRAB KPD 205 cells in a volume of 500 µL. Samples were incubated for 1.5 h at 60 °C and 37 °C, respectively and fixed in 2.5% glutaraldehyde in phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, MO, USA) and then postfixed in 1% osmium tetroxide in PBS. Following ethanol dehydration, samples were embedded in Epon resin, cut on a Leica UC7 ultramicrotome, and contrasted in uranyl acetate and lead citrate. Transmission electron microscopy studies were performed using an FEI Tecnai BioTwin Spirit microscope (FEI Company, Hillsboro, OR, USA).

LysC at a final concentration of 500 μg/mL was mixed with 10⁸ of S. aureus ATCC 25923 cells in a volume of 500 μL of 20 mM HEPES, pH 7.4. In the control reaction, equivalent volume of 20 mM HEPES, pH 7.4 was added to the bacterial cell suspension instead of the enzyme. Samples were incubated for 1.5 h at 37 °C, washed in phosphate buffer saline (PBS) (Sigma-Aldrich) and fixed overnight at 4 °C in 2.5% glutaraldehyde in PBS. Following rinsing, samples were post-fixed in 1% OsO₄ for 1 h at room temperature. After dehydration in a graded series of ethanol, probes were embedded in EPON resin, cut on an ultramicrotome Leica UC7, counterstained with uranyl acetate and lead citrate, and viewed with a FEI Tecnai BioTwin Spirit microscope (FEI Company, Hillsboro, Oregon, United States).