specimen collection, transport, and processing definitive diagnosis of brucellosis requires isolation of die organisms in cultures of blood, bone marrow, or other tissues. For the following reasons it is essential that the clinical microbiology laboratory be notified whenever brucellosis is suspected:
• To ensure that specimens are cultivated in an appropriate manner for optimum recovery from clinical specimens direct detection methods
Direct stains of clinical specimens are not particularly useful for the diagnosis of brucellosis. Preliminary studies using conventional and real-time polymerase chain reaction assays indicate that these assays may prove to be a reliable, sensitive, and specific means to directly detect Brucella spp. in clinical specimens.
Commercial blood culture systems, such as the BacT/ Alert, BACTEC, and lysis-centrifugation systems, have all successfully detected Brucella in blood.9 A recent comparison of the BACTEC 9240 with the Isolator blood culture systems found the BACTEC system more sensitive and faster in its detection of B. melitensis.11 Other blood culture botdes, such as those with brain-heart infusion and trypticase soy broth, also support the growth of brucellae if botdes are continuously vented and placed in a C02 incubator. Although the majority of isolates can be detected within 7 days using commercial systems, prolonged incubation of 30 days and periodic blind subcultures to blood and chocolate agar plates at 7, 14, and 30 days is recommended to maximize recovery of brucellae.10 Culture botdes may not become turbid. All subculture plates should be held for a minimum of 7 days.
Although Brucella spp. grow on blood and chocolate agars, supplemented media such as Brucella agar or some type of infusion base are recommended for specimen types other than blood. The addition of 5% heated horse or rabbit serum enhances growth on all media. Cultures should be incubated in 5% to 10% C02 in a humidified atmosphere; inoculated plates are incubated for 3 weeks before being discarded as negative.
On culture, colonies appear small, convex, smooth, translucent, nonhemolytic, and slighdy yellow and opalescent after at least 48 hours of incubation (Figure 38-1). The colonies may become brownish with age.
approach to identification
Presumptive identification of Brucella can be made, but appropriate biohazard facilities must be used. On Gram
Figure 38-1 Growth of Brucella spp. on chocolate agar after 2 days (A) and 4 days (B) of incubation.
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