Sheep s blood agar

Streptococcus pneumoniae E353, a capsule type 6 A human endophthalmitis strain, was kindly provided by Regis P. Kowalski (Charles T. Campbell Eye Microbiology Laboratory, University of Pittsburgh, Pittsburgh, Pennsylvania, USA). Frozen stocks of E353 were routinely cultured for isolation on sheep’s blood agar (Oxoid, Basingstoke, Hampshire, England) for 18–24 hours at 37 °C and 5% CO₂. Isolated colonies were inoculated into Todd Hewitt (Bacto^TM) broth containing 0.5% yeast extract (THY) and then incubated for approximately 18 hours at 37 °C and 5% CO₂. These 18-hour cultures were then diluted 100-fold in fresh THY and incubated until the optical density at 600 nm reached 0.23, which corresponded to approximately 1 × 10⁷ colony-forming units per mL (CFU/mL) as determined by previous growth curve analysis. Serial 10-fold dilutions of this logarithmic-phase subculture were prepared in sterile PBS (1.64 M NaCl: 2.3 × 10⁻³ M NaH₂PO₄: 7.7 × 10⁻³ M Na₂HPO₄, pH 7.0) and the third dilution (corresponding to a target of 10² CFU per 10 μL) was used for inoculation. Serial dilutions were also plated on blood agar to verify purity and quantity.