The tube with the PSB was vortexed vigorously. One hundred microliters of fluid, undiluted and diluted 1/100, was inoculated on two blood agar plates, and 100 ц.1 of undiluted fluid was inoculated on one hematin agar plate. The blood agar plates were incubated at 37°C in air and in anaerobic atmosphere, respectively. The hematin agar plate was incubated at 37°C in a C02-enriched atmosphere. The plates were observed for growth after one and two days. Recovery of ^lO cfu/ml of a particular pathogen was considered as significant growth.
Portions from BAL fluid were used for cultivation of Legionella, Mycobacteria, fungi, and viruses. Cultivation of Legionella was performed on the first portion of BAL with sterile water: BAL fluid without pretreatment and fluid decontaminated with KCl-HCl buffer was inoculated on BCYEa medium (buffered charcoal yeast extract agar with added a-ketoglutarate) and В MPA medium (BCYEa medium containing cefamandole, polymyxin B, and ani-somycin). The cultures were incubated at 37°C for five days. Fluid for culture of Mycobacteria was decontaminated with sodium lauryl sulphate and sodium hydroxide. After centrifugation, the pellet was neutralized with sulfuric acid and inoculated onto three drug-free Lowenstein-Jensen (LJ) culture tubes and one LJ culture tube containing 0.4 mg of isoniazid per liter. The cultures were incubated at 37°C in 5 percent CO* for one week, and thereafter at 37°C in air for nine weeks. Lavage fluid for culture of fungi was centrifuged and the pellet was inoculated on brain heart infusion (BHI) and Sabouraud agar. The agar plates were incubated at 20°C, 30°C, and 37°C for 14 days.
The BAL portions for virus isolation were treated with gentamicin (final concentration, approximately 300 mg/L). After centrifugation at 250 Xg for 10 min at 4°C, 0.1 to 0.2 ml of the supernatant was added to cell culture tubes. All specimens were inoculated in duplicate to three different cell lines: human foreskin fibroblasts, A549-cells (epithelioid cells originating from a human carcinoma), and monkey kidney cells (VERO or GMK). Cell cultures were observed for cytopathic effect every second day for ten days except for the fibroblasts that were inspected once a week for four weeks. Cytomegalovirus was identified by typical cytopathic effect in human fibroblasts. Herpes simplex virus isolates were confirmed and typed by immunofluorescence in the cell culture using monoclonal antibodies (Syva Co, Palo Alto, Calif).
Slides for microscopy were also prepared from BAL fluid after centrifugation. Pneumocystis carinii was detected with immunofluorescence (IF) technique using monoclonal antibodies (Merifluor, Meridian, Cincinnati). Acid-fast smear examination was performed after Ziehl-Neelsen staining. Cytologic preparations were examined after Papanicolaou and silver staining. Material from the NSB was used for microscopy of Gram-stained preparations.
Definition of Therapeutically Guiding Results
The patient charts were reviewed retrospectively. Frequently, empirical therapy was given while microbiologic results were pending. A positive microbiologic finding was defined as therapeutically guiding if the result changed the antimicrobial therapy or if a therapy was begun based on the result. It was also defined as therapeutically guiding if a previous treatment was continued based on the result of the bronchoscopy, and the recovery of this organism was conclusive, ie, recovery of P carinii, M tuberculosis, or Legionella species.