Modified Buffered Charcoal Agar Base is for isolation and cultivation of Legionella species

Legionella species are non-spore forming, narrow, gram-negative rods. Legionella causes pneumonia (Legionnaires disease) (1) or a milk, febrile disease (Pontiac fever). They do not oxidize or ferment carbohydrates in conventional media or grow on sheep blood agar. Growth is much better and more rapid on Buffered Charcoal Yeast Extract Agar (3, 4). Amino acids are the major sources of energy for Legionella . The amino acid L-cystine holds an absolute requirement as it plays major role in growth metabolism of Legionella (2). This amino acid as well as ferric pyrophosphate helps for the growth of Legionella. Modified Buffered Charcoal Agar is similar to Buffered Charcoal Yeast extract Agar Base except that the yeast extract is replaced by proteose peptone. This medium is recommended for isolation and cultivation of Legionella species from clinical and environmental specimens. The medium was formulated by Feeley et al (5) and Edelstein (7) modified it further. The media contains charcoal, which acts as a detoxicant. Proteose peptone acts as a rich source of vitamins, nitrogen as well as carbon. ACES Buffer maintains optimal pH for growth while L-cystine hydrochloride; ferric pyrophosphate and a-Ketoglutarate stimulate growth of Legionella species. For selective isolation, antibiotic supplements can be used to suppress contaminating microorganisms. Legionella Selective Supplement II (CCVC) (FD037) containing cephalothin, colistin, vancomycin and cycloheximide (8) or Legionella Selective Supplement IV (MWY) (FD040) containing glycine, polymyxin B, anisomycin, vancomycin, bromothymol blue and bromocresol purple (6) are often used. Wear gown, mask and gloves while handling Legionella cultures. Work in a safety hood. Directions: Suspend 20 grams in 500 ml distilled water. Heat to boiling to dissolve the medium completely. Sterilize by autoclaving at 15lbs pressure (121°C) for 15 minutes. Cool to 50°C and aseptically add sterile rehydrated contents of one vial of Legionella Supplement (FD041A and FD040). Mix well and pour into sterile Petri plates with constant agitation to ensure that charcoal particles are evenly distributed. For additional selectivity , Legionella Selective Supplements (FD017, FD037 and FD038) may be added.