Murine model of pneumococcal pneumonia.

S. pneumoniae serotype 3, 6303 (American Type Culture Collection, Manassas, VA) and the D39 strain of S. pneumoniae serotype 2 were grown to mid-log phase in Todd Hewitt broth (THB) with 0.5% yeast extract (Difco, Detroit, MI), washed in PBS, and serially diluted in sterile PBS. After anesthesia with ketamine (80 mg/kg) and xylazine (10 mg/kg) delivered via an intraperitoneal injection, a midline incision was made to expose the trachea, a 30-μl inoculum containing 50,000 colony forming units (CFUs) of S. pneumoniae was administered via the trachea with a 26-gauge needle, and the wound was closed with surgical glue (Vetbond, 3M, St. Paul, MN). Animals were observed for survival. In a separate group of mice, lung and spleen homogenates were prepared from mice harvested 24 h and 48 h (24 h only for D39 strain of S. pneumoniae) after infection following euthanasia with CO₂ asphyxiation. Serial dilutions of tissue homogenates were plated on blood agar, and CFUs were calculated as previously described (33).