**4.1. Culture**

The isolation of the etiological agent is the gold standard for pertussis diagnosis. To perform the bacterial isolation, a clinical sample from the nasopharynx should be obtained by aspiration or swabs. Aspirates give better yields than nasopharyngeal swabs though this last could be used, but swabs should be composed of Dacron or nylon if both culture and PCR are to be performed. While cotton swabs are not recommended since they contain substances that could inhibit *B. pertussis* growth, calcium alginate swabs are appropriate only for culture because they inhibit PCRs [44]. Successful recovery of the causative agent depends on a number of factors, including collection and transport conditions of the sample, the stage of disease in which the sample is collected, and the use of antibiotics. *B .pertussis* should be cultivated in Regan Lowe medium and/or Bordet Gengou agar supplemented with defibrinated blood in concentration of 7–15% (**Figure 1**). Addition of the antibiotic cephalexin has been recommended to inhibit growth of contaminating bacteria. However, since cephalexin has been suggested to also inhibit growth of *B. holmesii* [45], plates with and without cephalexin should be used. Incubation periods of up to 10–14 days are recommended for optimal sensitivity. Though *B*. *pertussis* growth may be retarded, *B*. *bronchiseptica* usually grows faster (1–3 days), and *B*. *parapertussis* shows an intermediate growth rate. Growth should be checked daily to prevent overgrowth by contaminating microorganisms.

After growth, *Bordetella* can be detected by Gram staining and identified by biochemical reactions, agglutination with specific sera or PCR. *Bordetella* species can be distinguished biochemically by oxidase, urease, motility, and nitrate reduction.
