TPC and BAL samples were processed within 20 min of collection according to the procedure described by Winterbauer et al. Briefly, after vortexing for 60 s both samples were serially diluted by factors of 10, 100, and 1,000, then plated in blood agar and chocolate agar media, and incubated in aerobiosis and in microaerophilia. Moreover, 0.1 ml of the TPC specimens were incubated under anaero-biosis for 48 h. Culture plates were evaluated for growth at 12 to 24 h and the bacteria isolated were identified by standard laboratory techniques.
Since the brush samples yield approximately 0.001 ml of secretion, growth of one colony in the 100-fold dilution plate indicates 103 cfu/ml of the specimen and reflects a bacterial concentration of 10s cfu/ml in the bronchial secretions. Previous studies have established that 10е cfu is the minimal bacterial concentration usually present in infected tissue. Therefore, bacterial counts 10 cfu/ml for TPC cultures were considered positive to certify etiology. TPC cultures below 10 cfu/ml were judged as “doubtful” and negative cultures were considered “not diagnostic.”
The expression “normal flora” was used when a mixed flora composed of less than 103 cfu/ml of a-hemolytic Streptococcus, Neisseria catarrhalis, or Staphylococcus epidermidis was isolated in BAL samples or sputum. When a single strain was isolated in a pure culture, the organism was registered as present independently of the colony count.
In parallel and in order to determine the extent of BAL contamination with oropharyngeal bacteria, quantitative cultures of BAL samples were obtained from 13 patients subjected to bronchoscopy for diagnosis of pulmonary infiltrates (diffuse interstitial fibrosis, 9; suspected tuberculosis, 3; and chronic eosinophilic pneumonia, 1). These patients were considered as a control group.