Brain heart infusion bhi agar 20 plates BD Difco BBL

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	Isolates were cultured in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB plus 2% (v/v) lysed horse blood, Brain-Heart-Infusion (BHI) (Becton Dickinson, Heidelberg, Germany) and Caso broth (Sigma-Aldrich, Seelze, Germany), which are frequently used media for the cultivation of Bordetella spp. [17–19]. Incubation was performed at 35°C in ambient air. Sampling was performed every two hours during the first 26 hours and, thereafter every six hours.

Protocol tips

Isolates were cultured in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB plus 2% (v/v) lysed horse blood, Brain-Heart-Infusion (BHI) (Becton Dickinson, Heidelberg, Germany) and Caso broth (Sigma-Aldrich, Seelze, Germany), which are frequently used media for the cultivation of Bordetella spp. [17–19]. Incubation was performed at 35°C in ambient air. Sampling was performed every two hours during the first 26 hours and, thereafter every six hours.

Publication protocol

Isolates were cultured in cation-adjusted Mueller-Hinton broth (CAMHB), CAMHB plus 2% (v/v) lysed horse blood, Brain-Heart-Infusion (BHI) (Becton Dickinson, Heidelberg, Germany) and Caso broth (Sigma-Aldrich, Seelze, Germany), which are frequently used media for the cultivation of Bordetella spp. [17–19]. Incubation was performed at 35°C in ambient air. Sampling was performed every two hours during the first 26 hours and, thereafter every six hours. At each sampling time, starting at hour 0, a 10-fold serial dilution in 0.9% saline was done for every isolate, and four different dilutions were plated in duplicate on Columbia sheep blood agar. Enumeration plates were incubated at 35°C in ambient air. To calculate the average of colony forming units, two dilutions between 5 and approximately 200 cfu/mL were counted from the plated dilutions and the mean value of the two dilutions was determined. All experiments were performed three times independently.

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