Samples were collected from the eyelids, the upper eyelashes, and the conjunctival sacs of all subjects, the contact lenses and the lens cases of ortho-k wearers, and the spectacle frames of the control group. For the conjunctival sac, a sterile swab soaked in sterile phosphate-buffered saline (PBS) was used to swab the lower conjunctiva of the left eye with a rolling motion from the lateral cantus toward the medial cantus. For the eyelid and eyelashes, two sterile cotton swabs moistened in sterile PBS were used to swab the central part of the lower eyelid and the eyelashes individually. The contact lens worn on the left eye was removed by the subject per normal routine and placed immediately in a new sterile lens case containing 2 mL sterile PBS, and the case was brought to the clinic for analysis. The subjects also brought their existing lens cases (empty) for microbial assessment. The lens case containing the lens was vortexed vigorously for 30 seconds to loosen any micro-organisms adhering to the lens surface. The lens was then removed from the case using sterile forceps and returned to the subject. The lens extract was poured into a bijou bottle containing 10 mL sterile brain heart infusion broth (BHI broth). For examination of flora from the routinely used lens case, a cotton swab soaked in Dey-Engley neutralizing broth (Oxoid, Basingstoke, UK) with 2 mM EDTA was used to sample the inner surfaces and the screw tops of the lens case; EDTA is used to help release bacteria from biofilms that may be present on the lens case surface. At this low concentration, EDTA is not bactericidal
15,16 and has been used in previous studies of contamination of lens cases.
17,18 For the spectacle frame, a sterile cotton swab moistened in sterile PBS was used to sample the right nose pad surface and the right arm of the spectacle frame. Each swab was broken off in a bijou bottle containing 10 mL sterile BHI broth and then vortexed for 30 seconds to release micro-organisms. After 16-hour incubation at 37°C, the broths were subcultured on SaSelect agar (Bio-Rad, Redmond, WA, USA) and incubated for 24 hours. For cultures showing growth, colonies displaying typical staphylococcal morphology were isolated, Gram stained, and identified using catalase, tube coagulase test, and Staphaurex Plus test (Murex Biotech Ltd, Dartford, UK). Strains positive for tube coagulase test and Staphaurex Plus test were reported as
S. aureus, while those negative for these tests were reported as CNS. For the conjunctival sacs, both
S. aureus and CNS were characterized, as both may be a cause of infection at this site. For all other samples only
S. aureus, which is a more pathogenic organism, was investigated.