Legionella Culture (Rule out)


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Subject: Legionella Culture (Rule out)

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  • This test is requested for diagnosis of legionellosis by culture of patient specimens, usually of the lower respiratory tract. Testing is recommended on patients with pneumonia who fail treatment, have severe pneumonia, are immunocompromised, or have epidemiologic risk for legionellosis. Special testing, usually performed outside of clinical microbiology laboratories, is needed for evaluation of environmental cultures for isolation of Legionella species.

Special Collection and Transport Instructions

  • Specimens should be submitted early in the acute phase of infection.

  • Sputum (expectorated or induced), BAL, bronchial brush, lung biopsy, or tracheal aspirate specimens are usually submitted for culture to rule out Legionella.

  • Submission of multiple specimens is recommended to improve sensitivity of detection because shedding may be intermittent for this intracellular pathogen.

  • Blood, cardiac valve, or other specimen types are occasionally submitted when extrapulmonary legionellosis is suspected. (If Legionella endocarditis is suspected, alert the laboratory because special processing and culture techniques are required.)


  • Culture Method:

    • All specimens should be inoculated onto supplemented BCYE agar, both nonselective and selective.

    • Specimens may be diluted 1:10 in tryptic soy broth to prepare culture inoculum. For specimens that are likely to be heavily contaminated by endogenous flora, a 1:10 dilution of specimen in 0.2M KCl acid wash (pH = 2.2) is recommended to improve isolation of Legionella. The specimen is incubated at room temperature for 4 minutes, and then aliquots are inoculated onto selective and nonselective BCYE media as for unwashed specimens.

    • Cultures are incubated at 35–37°C in a humidified incubator. CO2 supplementation (2–5%) may be used.

  • Turnaround time: Cultures are inspected for up to 5 days after inoculation. Additional time is required after isolation for confirmation and further characterization.


  • Expected results: Negative.

  • Positive results: Positive cultures confirm a diagnosis of legionellosis. Isolates from Legionella cultures must be confirmed as Legionella species by further testing and characterization.

  • Negative results: Because Legionella may be shed intermittently, a negative culture does not rule out legionellosis.


  • Legionella are typically present in low concentrations in patient specimens. Isolation of Legionella from infected extrapulmonary specimens is inconsistent.

  • Diagnosis of legionellosis may require multiple testing modalities, including culture, serology, PCR, and antigen detection methods, in conjunction with clinical findings.

  • Common pitfalls:

    • The rejection criteria applied to sputum specimens for routine bacterial cultures should not be applied to specimens submitted for Legionella culture.

    • Legionella may be present in very low concentrations in respiratory secretions. Therefore, BAL and bronchial brush specimens should be directly inoculated onto BCYE media before dilutions are prepared for quantitative bacterial cultures.