Fig 3.

Removal of EDTA or EDTA saturation with divalent cations resumes biofilm growth. (a) Measurement of biofilm growth by time-lapse microscopy. (i) Two independent sets of C. neoformans biofilms were grown in inducing medium for 9 h. (ii) Cells were subsequently incubated with medium containing 0.025 mM EDTA or medium alone for an additional 24 h. (iii) Cells were thoroughly washed to remove EDTA and grown again in EDTA-free inducing medium for another 9 h. Biofilm formation was recorded by using time-lapse microscopy, the areas of three biofilm microcolonies were measured, and the averages were plotted (error bars indicate standard errors of the means). The asterisk indicates that untreated cells had grown to confluence and, therefore, that individual microcolonies could not be measured after this stage. (b) Measurement of biofilm growth by XTT assays. Biofilms were grown for 9 h in inducing medium and then incubated with 0.025 mM EDTA for 24 h. Various concentrations of Mg²⁺ and/or Ca²⁺ (0 to 1 M) were added to fresh EDTA-containing inducing medium, and cells were incubated for an additional 24 h. XTT viability assays were used to measure biofilm growth. The averages of data from three wells were plotted, and standard deviations are shown. Controls consisting of untreated biofilms (C. neoformans only [Cn]), biofilms treated with 0.025 mM EDTA (Cn + EDTA), and EDTA-treated biofilms washed and subsequently grown in EDTA-free medium (Cn + EDTA + media) were included.