LEGIONELLA - World Health Organization

30 minutes (Maiwald, Helbig & Lück, 1998), and by treating with acid (Bopp et al., 1981;
ISO, 1998; ISO 2004). If using an acid treatment, an acid buffer of pH 2.2 should be used for
five minutes, although this may also inhibit the growth of legionellae (Lück, Helbig &
Schuppler, 2002). Homogenates of sputa and tissues, and centrifuged deposits from more fluid
clinical specimens should be cultured directly, and after treating with heat or an acid buffer
(Stout, Rihs & Yu, 2003).
Although it may be possible to isolate legionellae on the non-selective growth medium BCYE
(particularly from clinical specimens), it is usually necessary to use modified versions of
BCYE containing an antibiotic supplement to suppress the background flora, such as:
• polymixin, anisomycin and cefamandole (Edelstein, 1981)
• glycine, vancomycin and polymixin, plus one of the following:
– cycloheximide (Dennis, 1988b)
– natamycin, which is an alternative antifungal to cycloheximide that is less toxic to
humans (Edelstein & Edelstein, 1996).
An alternative medium — Wadowsky and Yee medium (MWY) as modified by Edelstein
(1982b) — includes vancomycin, bromothymol blue and bromocresol purple, and is used to
increase the differentiation of legionellae from the background organisms (Wadowsky & Yee,
1981; Vickers et al., 1981). Morrill et al. (1990) advocated the additional use of albumin to
increase the recovery of L. micdadei and L. bozemanii.
In environmental investigations of outbreaks of legionellosis, culture has been used to detect
legionellae in the environment. As a result, most of the epidemiologically relevant information
concerning legionellosis is based on direct culture data. All agar plates are inoculated with a
portion of sample (generally 0.1–0.2 ml) by the spread plate technique and incubated at
36 °C, preferably in a humidified 2.5% carbon dioxide (CO 2 ) atmosphere or candle
extinction jar.
11.4.6 Interpreting results
To date, no direct relationship has been established between the risk of infection and the
number of Legionella detected in a water system using the generally adopted culture method.
Recovery of L. pneumophila by culture is poor because:
• Legionella exist with other background heterotrophic bacteria; therefore, the sample needs
to be treated with heat or acid to repress the growth of non-Legionella bacteria on the
culture media
• antibiotics need to be added to the culture medium for Legionella growth.
LEGIONELLA AND THE PREVENTION OF LEGIONELLOSIS