Bbl trypticase soy agar tsa 2 with sheep blood

Streptococcus pneumoniae infections were performed as previously described.(Boe, Nelson, Zhang, Quinton, & Bagby, 2003 (link); Siggins et al., 2011 (link)) Briefly, S. pneumoniae (ATCC® BAA-334™, strain TIGR4 [JNR.7/87], Capsular serotype 4; American Type Culture Collection, Manassas, VA) were grown in 100 mL Todd Hewitt Broth (Becton Dickinson, Franklin Lakes, NJ) in a CO₂ incubator (5% CO₂) at 37^oC for 6 hours. Bacteria were then pelleted by centrifugation (2,000 x g for 15 minutes at 4 ^oC), washed twice with phosphate-buffered saline (PBS), and resuspended in PBS at a concentration of 1 × 10⁵ colony-forming units (CFU)/mL. The actual number of viable bacteria were determined by serial dilutions onto BD BBL™ Trypticase™ Soy Agar (TSA II™) with Sheep Blood and performing standard colony counts. Twenty-four hours after the final ethanol binge, animals were anesthetized with isoflurane and given 1 × 10⁵ CFU bacteria in 50 μL PBS via intranasal (i.n.) administration. Animals were allowed to recover from anesthesia and returned to their cages. Mice were sacrificed 48 hrs. post infection. S. pneumoniae burden was then determined by serial dilutions onto BD BBL™ Trypticase™ Soy Agar (TSA II™) with Sheep Blood and performing standard colony counts.