Charcoal-yeast extract agar is a new bacteriological medium that supports excellent growth of the Legionella pneumophila. It results from modifications made in an existing L. pneumophila medium, F-G agar. Yeast extract, instead of an acid hydrolysate of casein, serves as the protein source. Beef extractives and starch are not added. Activated charcoal (Norit A or Norit SG) is included at 0.20% (wt/vol). Comparison of charcoal-yeast extract and F-G agars showed that a greater number of colony-forming units of L. pneumophila was recovered from a standardized tissue inoculum on charcoal-yeast extract agar (4.35 x 106 colonyforning units) than on F-G agar (4.85 x 104 colony-forming units). Macroscopic colonies of L. pneumophila were visible on the new medium within 3 days, whereas 4 days of growth was required on F-G agar. McDade et al. initially isolated the Legionnaires disease bacterium, now named Legionella pneumophila, by using guinea pigs and embryonated chicken eggs (2, 9). For the purpose of growing L. pneumophila on artificial media, Weaver inoculated 17 different bacteriological agars with an L. pneumophila-infected yolk sac suspension. Mueller-Hinton agar supplemented with 1% hemoglobin and 1% IsoVitaleX (BBL

Pittsburgh pneumonia agent (PPA) was recently cultivated from infected egg yold on charcoal yeast extract agar. PPA has now been isolated both from infected egg yolk and human lung tissue on charcoal yeast extract agar and on a new medium, buffered charcoal yeast extract agar. PPA resembles Legionella pneumophila and other Legionella-like organisms in requirements for growth and composition of fatty acids. It differs in genetic relatedness, antigenic composition, and colonial morphology and has distinctive characteristics that allow it to be identified. The name Legionella pittsburgensis species nova is proposed for this organism.

At the site of a legionellosis outbreak, amoebae and two ciliates, Tetrahymena sp. and Cyclidium sp., were isolated from cooling-tower water containing Legionella pneumophila. The Tetrahymena sp. and the amoebae repeatedly showed the ability to support intracellular multiplication of L. pneumophila. Both were isolated from cooling towers specifically implicated as the source for the spread of legionellosis. These protozoa may be reservoirs supporting the survival and multiplication of virulent legionellae in cooling-tower water.