Direct in-vitro isolation of the Legionnaires' disease bacterium in two fatal cases. Cultural and staining characteristics.

Two cases of Legionnaires' disease were diagnosed by direct isolation of the organism, from pleural fluid obtained before death in one case and lung tissue obtained after death in both cases. The organisms were recovered on a commercially prepared, enriched chocolate agar (Gibco, Madison, Wisconsin). Subcultures grew on commercially prepared, enriched chocolate agar (Baltimore Biological Laboratories, Cockeysville, Maryland) and on in-house enriched chocolate agar prepared with GC Medium Base (Difco, Detroit, Michigan). No growth was obtained on enriched chocolate agar prepared with trypticase soy agar. The organisms were poorly visualized in Gram-stained sections of formalin-fixed lung tissue. In Giemsa-stained sections poorly stained intracellular and extracellular slender rods were seen. However, with a silver impregnation stain, either a modified Dieterle or a modified Warthin-Starry procedure, many large, blunt-ended rods were seen. Smears prepared from minced formalin-fixed lung tissue and stained with a fluorescent antibody conjugate contained large numbers of well-stained organisms.