Streptococcus Pneumoniae suspension preparation

Streptococcus Pneumoniae suspension preparation S. Pneumoniae capsular type 6A (DBL6A) was previously described as a virulent strain (Briles et al., 1992). Hemoculture of mouse second passage isolates were provided by Dr. Denis Martin (ID Biomedical, Quebec, Canada) and a suspension of the provided culture in Todd Hewitt medium supplemented with 0.5% yeast extract (THY) was incubated at 37 o C x 8% CO2 until reaching an optical density (O.D.600nm) of 0.4. Thereafter, 500 ml of the suspension [8 x 10 7 colony forming units (C.F.U.)] was injected i.p. in Balb/c male mice. In the next day, mice were anesthetized for cardiac puncture and sacrifice. The blood drawn via cardiac puncture was supplemented with glycerol (20%) and frozen at -80 o C. In the day of the infection, an 50 ml aliquot of the cardiac puncture was suspended in 5 ml of THY medium and incubated as described. This preparation was serially diluted until a working suspension of 50 -100 C.F.U./ml. For dose verification, 100 ml of the suspension was plated on bloodagar from two different sources (Fisher Scientific and agar plates provided by a local microbiology laboratory).