Isolation and Identification of Legionella

Two respiratory specimens were collected from each patient: one sample inoculated onto the culture media and the other stored at −70°C until molecular assay. Sputum specimens were decontaminated by heat treatment (56°C for 30 minutes), and BAL samples were concentrated by centrifugation. The prepared specimens were cultured onto an unselective medium – buffered charcoal–yeast extract (BCYE) agar (Oxoid, Basingstoke, UK) – and a selective medium, modified Wadowsky–Yee agar (Oxoid) supplemented with l-cysteine. The plates were incubated in candle jars (3%–5% CO₂) at 35°C in a humidified atmosphere and inspected for 4–14 days for the presence of Legionella spp. colonies. Colonies showing characteristics of Legionella spp., such as grayish-white, shiny colonies were selected. Gram staining was done to show the thin, faintly stained filamentous Gram-negative morphology. Suspected colonies were subcultured on BCYE agar with and without l-cysteine and unselective media, such as sheep-blood agar and MacConkey agar, for verification. Isolates that grew on BCYE agar with l-cysteine, but not on the other media, were considered Legionella. Strains unable to grow on media without l-cysteine were further identified by PCR and LAMP for the mip gene.14 (link)