Transmission electron microscopy protocols for capsule visualisation in pathogenic respiratory and meningeal bacteria

The human nasopharynx has a commensal bacterial flora which may become pathogenic (invasive), causing pneumonia, sepsis, meningitis and otitis media. A major virulence factor in this pathogenesis is the presence of a polysaccharide bacterial capsule, the production of which is controlled by a number of interacting variables (including temperature and capsule gene presence/expression/phase variation). Capsule composition is the basis of routine microbiological typing as well as the target of current pneumococcal vaccines. However, there is growing evidence that strains lacking a capsule (non-typeable strains) may also cause disease. Several different transmission electron microscopy protocols were tested on control cultures (known phenotypes) in order to visualise bacterial capsular material, ultimately for the phenotypic description of routinely grown, invasive, non-typeable isolates from the South African national laboratory-based, surveillance programme. Of these protocols, the most successful involved the use of ruthenium red, L-lysine acetate and an osmotically-adjusted buffer, though for some Gram-negative bacterial cells (those lacking sialic acid/heteropolymeric polysaccharides in their cell walls), it proved necessary to use a similar protocol but with the ruthenium red being replaced with another cationic dye, namely alcian blue (pyridine variant). The pyridine variant of alcian blue stained better than alcian blue 8XG, albeit somewhat indiscriminately (in that it was not specific for capsular polysaccharides). Identification and characterisation of the capsular status of these sorts of Gram-negative cells therefore need careful analysis of phenotypic information, and correlation with both genotypic data and environmental variables.