Saccharomyces cerevisiae invsc1

A biological assay was designed to verify that tested antimicrobials were penetrating the contact lens and retained functionality. The contact lens diffusion model was performed as described previously (Figure 1); however, at 10 minute intervals over a 1 hour period, 10 μl aliquots of the with diffused antibiotic were spotted onto lawns of susceptible test organisms. In this assay Staphylococcus epidermidis strain RP62A was used for moxifloxacin and PHMB, and Saccharomyces cerevisiae InvSc1 (Invitrogen) was used for amphotericin B. Microbial cultures were grown overnight in Tryptic Soya Broth (TSB) or yeast extract, peptone, and dextrose broth (YPD). Five microliters of overnight culture was added to 150 μl of PBS and plated onto nutrient rich agar plates as a lawn and dried briefly before addition of the test aliquots. The plates were incubated overnight at 37°C for S. epidermidis and 30°C for S. cerevisiae. Zones of growth inhibition were visually assessed. Prevention of microbial growth indicated that the antimicrobial was capable of diffusing through the SH contact lens applied directly onto the lawns. The sensitivity of the assay was established using a 2-fold serial dilution series of the test antimicrobials. The limit of detection was 4.88 μg/ml for moxifloxacin, 48.8 μg/ml for PHMB, and 39.1 μg/ml for amphotericin B.