In vitro growth and analysis of Candida biofilms

Evaluation of fungal biofilm formation can be performed using several techniques. In this protocol, we describe methods used to form Candida biofilms on three different medical device substrates (denture strips, catheter disks and contact lenses) to quantify them and to evaluate their architecture and drug susceptibility. Biofilm formation involves adhesion of fungal cells to pretreated substrates, followed by growth in medium. Formed biofilms are quantified by determining their metabolic activity and dry weight, whereas their gross morphology and architecture are evaluated using fluorescence microscopy, scanning electron microscopy and confocal scanning laser microscopy techniques. Susceptibility of biofilms is determined by comparing their metabolic activity in the presence of antifungal agents with that in their absence. The methods described here can be completed in a typical laboratory with minimum involvement of software. Evaluation of the growth of fungal biofilms and their analyses can be completed using the described methods in approximately 15 d.