Penetration of antifungal agents through Candida biofilms

A filter disk assay is described, which measures the penetration of antifungal agents through Candida biofilms. The technique involves forming a colony biofilm on a polycarbonate membrane filter, and capping it with a second, smaller membrane filter followed by a wetted paper disk of the type used in zone-of-inhibition assays. The entire assembly is transferred to agar medium containing the antifungal agent of interest. During subsequent incubation, the drug diffuses out of the agar and through the biofilm 'sandwich' to the moistened paper disk. The drug concentration in the disk can be determined by measuring the zone of growth inhibition that it produces on medium seeded with an indicator strain of Candida albicans in standard bioassays. Additional procedures are outlined for determining the viabilities of drug-treated biofilms and for examining biofilm morphology by scanning electron microscopy.