Overnight cultures of either S. aureus strain 8325-4 or S. epidermidis strain 14990 were diluted 1:100 in sterile tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) and incubated at 37°C. Bacteria were grown to an absorbance of 0.3 to 0.42 at 650 nm, and cultures were serially diluted in sterile TSB to approximately 1 × 105 CFU/mL (1000 CFU inocula) or 5 × 107 CFU/mL (500,000 CFU inocula).
For injection of bacteria into the anterior chamber, rabbits were anesthetized by subcutaneous injection of a 1:5 mixture of 100 mg/mL xylazine (Rompun; Miles Laboratories, Shawnee Mission, KS) and 100 mg/mL ketamine HCl (Ketaset; Bristol Laboratories, Syracuse, NY). One drop of proparacaine HCl (0.5%; Falcon Pharmaceuticals, Fort Worth, TX) was administered to each eye before injection. A 30-gauge needle attached to a syringe (Hamilton Co., Reno, NV) was inserted through the cornea into the anterior chamber of anesthetized rabbits, to inject each eye with 10 μL of either S. aureus or S. epidermidis (approximately 1,000 CFU or 500,000 CFU). Serial dilutions of bacterial cultures were made in sterile TSB, and 0.1-mL aliquots were plated onto tryptic soy agar (TSA) in triplicate to confirm the number of CFU injected. At various times (1, 5, 10, 15, 20, and 25 hours) postinfection (PI), rabbits were killed by intravenous injection with sodium pentobarbital solution (100 mg/mL; Sigma-Aldrich, St. Louis, MO). Immediately before death, a 26-gauge needle on a 1-mL syringe was inserted through the cornea into the anterior chamber of anesthetized rabbits, and an aliquot (0.1 mL) of aqueous humor was removed. Because of the small sample size of aqueous humor per eye, undiluted aqueous humor samples could not be cultured. Aqueous humor samples were serially diluted in sterile phosphate buffered solution (PBS), aliquots (0.1 mL) were inoculated onto TSA plates, and the plates were incubated at 37°C for 24 hours. Colonies were counted, and CFU per anterior chamber were expressed as base 10 logarithms.