Infected cells, or pieces of infected lungs were washed with sodium cacodylate buffer (pH, 7.4) and fixed in 3% glutaraldehyde. Cells were then osmicated and dehydrated prior to embedding in EPON 812 resin36 (link). Polymerized blocks were cut into 80–100 mm ultrathin sections and collected on 200 or 400 mesh size copper grids. Sections were stained with Uranyl acetate (10 min–1 hr), followed by Reynolds lead citrate (2 min) and then imaged under 80 and 100 KeV operating voltages of a Tecnai 12 Biotwin transmission electron microscope (FEI Co, The Netherlands). Images were captured using a side mounted 2 k × 2k CCD camera (Megaview III, SIS Germany). Size calibration and morphometry was done using the TIA image analysis software (FEI Co, The Netherlands).
Extensive control imaging was done to ensure that the images collected were free of artifacts. This included monitoring for the natural preservation of nucleus, nuclear envelope, mitochondria and other cyto-organelles. Further, an adequate number of cell profiles and imaging fields were seen before any interpretation was made. Finally, we also ensured that our interpretations were based on published literature and standard reference material on ultrastructural pathology. For bacterial detection and enumeration, only those displaying morphologically relevant features as described in the literature were considered.
Extensive control imaging was done to ensure that the images collected were free of artifacts. This included monitoring for the natural preservation of nucleus, nuclear envelope, mitochondria and other cyto-organelles. Further, an adequate number of cell profiles and imaging fields were seen before any interpretation was made. Finally, we also ensured that our interpretations were based on published literature and standard reference material on ultrastructural pathology. For bacterial detection and enumeration, only those displaying morphologically relevant features as described in the literature were considered.