Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) and transmission electron micrography (TEM) of infected lung tissue.

Serial sections from an infected segment (DR12, LCD) confirming the identity of the presumed bacterial microcolonies growing in the agar beads. The haematoxylin and eosin stained section (a) has at its centre, a discrete circular bead containing one presumed cigar-shaped bacterial colony (*), and partially surrounded by another predominantly circular bead structure containing a further four distinct presumed colonies (∧). The peripheral margin of the coalesced bead matrix is surrounded by a halo of neutrophils (▴) which appears to infiltrate towards the centre from the lower right quadrant. The PNA FISH image (B) is from a section serial to (A), and confirms that the presumed colonies are indeed made up of Pseudomonas aeruginosa bacteria (bright orange stain). TEM images confirmed that the predominant cell type surrounding beads was the neutrophil. In many instances these cells had a highly ruffled surface architecture of microvillus-like projections (C) (bar  = 10 µm). Only occasionally were single bacteria found separate from the agar bead matrix environment, and occasionally found in blood or lymphatic vasculature (D, *) (bar  = 2 µm). Macrophages showed evidence of bacterial engulfment (E) (bar  = 2 µm).